CYR61 compositions and methods

ABSTRACT

Polynucleotides encoding mammalian ECM signaling molecules affecting the cell adhesion, migration, and proliferation activities characterizing such complex biological processes as angiogenesis, chondrogenesis, and oncogenesis, are provided. The polynucleotide compositions include DNAs and RNAs comprising part, or all, of an ECM signaling molecule coding sequence, or biological equivalents. Polypeptide compositions are also provided. The polypeptide compositions comprise mammalian ECM signaling molecules, peptide fragments, inhibitory peptides capable of interacting with receptors for ECM signaling molecules, and antibody products recognizing Cyr61. Also provided are methods for producing mammalian ECM signaling molecules. Further provided are methods for using mammalian ECM signaling molecules to screen for, and/or modulate, conditions and disorders associated with angiogenesis, chondrogensis, and oncogenesis; ex vivo methods for using mammalian ECM signaling molecules to prepare blood products are also provided. Additionally, modulators, such as peptide modulators, of an ECM signaling molecule activity are provided. Further provided are methods for screening for modulators of a Cyr61 polypeptide-integrin receptor inteaction, as well as methods of treating conditions and disorders associated with such an interaction.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is the 371 National Stage of International ApplicationNo. PCT/US01/03267, filed Jan. 31, 2001, which claims the benefit ofU.S. Provisional Application No. 60/204,364, filed May 15, 2000 and U.S.Provisional Application No. 60/238,705, filed Oct. 6, 2000 and which isa continuation-in-part of U.S. application Ser. No. 09/495,448, filedJan. 31, 2000 (now U.S. Pat. No. 6,790,606), which is acontinuation-in-part of U.S. application Ser. No. 09/142,569, filed Apr.2, 1999 (now U.S. Pat. No. 6,413,735), which is the 371 National Stageof International Application No. PCT/US97/04193, filed Mar. 14, 1997 andwhich claims the benefit of U.S. Provisional Application No. 60/013,958,filed Mar. 15, 1996.

FIELD OF THE INVENTION

The present invention is directed to materials and methods involvingextracellular matrix signaling molecules in the form of polypeptidesinvolved in cellular responses to growth factors. More particularly, theinvention is directed to Cyr61-, Fisp12-, and CTGF-relatedpolynucleotides, polypeptides, compositions thereof, methods ofpurifying these polypeptides, and methods of using these polypeptides.

BACKGROUND OF THE INVENTION

The growth of mammalian cells istightlyregulated bypolypeptide growthfactors. In the adult animal, most cells are metabolically active butare quiescent with regard to cell division. Under certain conditions,these cells can be stimulated to reenter the cell cycle and divide. Asquiescent cells reenter the active growth and division phases of thecell cycle, a number of specific genes, the immediate early genes, arerapidly activated. Reentry to the active cell cycle is by necessitytightly regulated, since a breakdown of this control can result inuncontrolled growth, frequently recognized as cancer. Controlled reentryof particular cells into the growth phase is essential for suchbiological processes as angiogenesis (e.g., blood vessel growth andrepair), chondrogenesis (e.g., skeletal development and prosthesisintegration), oncogenesis (e.g., cancer cell metastasis and tumorneovascularization), and other growth-requiring processes.

Angiogenesis, the formation of new blood vessels from the endothelialcells of preexisting blood vessels, is a complex process which involvesa changing profile of endothelial cell gene expression, associated withcell migration, proliferation, and differentiation. Angiogenesis beginswith localized breakdown of the basement membrane of the parent vessel.In vivo, basement membranes (primarily composed of laminin, collagentype IV, nidogen/entactin, and proteoglycan) support the endothelialcells and provide a barrier separating these cells from the underlyingstroma. The basement membrane also affects a variety of biologicalactivities including cell adhesion, migration, and growth duringdevelopment and differentiation.

Following breakdown of the basement membrane, endothelial cells migrateaway from the parent vessel into the interstitial extracellilar matrix(ECM), at least partially due to chemoattractant gradients. Themigrating endothelial cells form a capillary sprout, which elongates.This elongation is the result of migration and proliferation of cells inthe sprout. Cells located in the leading capillary tip migrate towardthe angiogenic stimulus, but neither synthesize DNA nor divide.Meanwhile, behind these leading tip cells, other endothelial cellsundergo rapid proliferation to ensure an adequate supply of endothielialcells for formation of the new vessel. Capillary sprouts then branch attheir tips, the branches anastomose or join with one another to form alumen, the basement membrane is reconstituted, and a vascular connectionis established leading to blood flow.

Alterations in at least three endothelial cell functions occur duringangiogenesis: 1) modulations of interactions with the ECM, which requirealterations of cell-matrix contacts and the production ofmatrix-degrading proteolytic enzymes; 2) an initial increase andsubsequent decrease in endothelial cell migration, effecting celltranslocation towards an angiogenic stimulus; and 3) a transientincrease in cell proliferation, providing cells for the growing andelongating vessel, with a subsequent return to the quiescent cell stateonce the vessel is formed These three functions are realized byadhesive, chemotactic, and mitogenic interactions or responses,respectively. Therefore, control of angiogenesis requires interventionin three distinct cellular activities:. 1) cell adhesion, 2) cellmigration, and 3) cell proliferation. Another biological processinvolving a similar complex array of cellular activities ischondrogenesis.

Chondrogenesis is the cellular process responsible for skeletalorganization, including the development of bone and cartilage.Chondrogenesis, like angiogenesis, involves the controlled reentry ofquiescent cells into the growth phase of the cell cycle. The growthphase transition is associated with altered cell adhesioncharacteristics, changed patterns of cell migration, and transientlyincreased cell proliferation. Chondrogenesis involves the initialdevelopment of chondrogenic capacity (i.e., the proto-differentiatedstate) by primitive undifferentiated mesenchyme cells. This stageinvolves the production of chondrocyte-specific markers without theability to produce a typical cartilage ECM. Subsequently, the cellsdevelop the capacitytoproduce acartilage-specific ECM as theydifferentiate into chondrocytes. Langille, Microscop. Res. & Tech.28:455-469 (1994). Chondrocyte migration, adhesion, and proliferationthen contribute to the development of bony, and cartilaginous, skeleton.Abnormal elaboration of the programmed development of cellsparticipating in the process of chondrogenesis results in skeletaldefects presenting problems that range from cosmetic concerns tolife-threatening disorders.

Like angiogenesis and chondrogenesis, oncogenesis is characterized bychanges in cell adhesion, migration, and proliferation. Metastasizingcancer cells exhibit altered adhesion and migration properties.Establishment of tumorous masses requires increased cell proliferationand the elaboration of the cellular properties characteristic ofangiogenesis during the neovascularization of tumors.

Abnormal progression of angiogenesis orchondrogenesis, as well as mereprogression of oncogenesis, substantially impairs the quality of lifefor afflicted individuals and adds to modern health care costs. Thefeatures common to these complex biological processes, comprisingaltered cell adhesion, migration, and proliferation, suggest that agentscapable of influencing all three of these cellular activities would beeffective in screening for, and modulating, the aforementioned complexbiological processes. Although the art is aware of agents that influenceindividual cellular activities, e.g., integrins and selectins (celladhesion), chemokines (cell migration), and a variety of growth factorsor cytokines (cell proliferation), until recently no agent has beenidentified that exerts an influence over all three cellular activitiesin humans.

Murine Cyr61 (CYsteine-Rich protein) is a protein expressed in activelygrowing and dividing cells that may influence each of these threecellular activities. RNase protection analyses have shown that the geneencoding murine Cyr61, murine cyr61, is transcribed in the developingmouse embryo. O'Brien et al., Cell Growth & Diff. 3:645-654 (1992). Insitu hybridization analysis showed that expression of cyr61 during mouseembryogenesis is closely correlated with the differentiation ofmesenchymal cells, derived from ectoderm and mesoderm, intochondrocytes. In addition, cyr61 is expressed in the vessel walls of thedeveloping circulatory system. These observations indicate that murinecyr61 is expressed during cell proliferation and differentiation, whichare characteristics of expression ofgenes involved in regulatorycascades that control the cell growth cycle.

Further characterization of the Cyr61 polypeptide has been hampered byan inability to purify useful quantities of the protein. Efforts topurify Cyr61 in quantity by overexpression from either eukaryotic orprokaryotic cells typically fail. Yang, University of Illinois atChicago, Ph. D. Thesis (1993). One problem associated with attempting toobtain useful quantities of Cyr61 is the reduction in mammalian growthrates induced by overexpression of Cyr61. Another problem with Cyr61purification is that the cysteine-rich polypeptide, when expressed inbacterial cells using recombinant DNA techniques, is often found ininsoluble protein masses. Nevertheless, Cyr61 has been characterized asa polypeptide of 349 amino acids, containing 39 cysteine residues, ahydrophobic putative N-terminal signal sequence, and potential N-linkedglycosylation sites (Asn₂₈ and Asn₂₂₅). U.S. Pat. No. 5,408,040 atcolumn 3, lines 41-54, Grotendorst et al., incorporated herein byreference (the '040 Patent). Recently, proteins related to Cyr61 havebeen characterized. For example, a human protein, Connective TissueGrowth Factor (CTGF), has been identified. (See '040 Patent). CTGF isexpressed in actively growing cells such as fibroblasts and endothelialcells ('040 Patent, at column 5, lines 62-64), an expression patternshared by Cyr61. In terms of function, CTGF has been described as aprotein growth factor because its primary biological activity has beenalleged to be its mitogenicity ('040 Patent, at column 2, lines 25-27and 53-55). In addition, CTGF reportedly exhibits chemotactic activity.'040 Patent, at column 2, lines 56-59. In terms of structure, thepolynucleotide sequence encoding CTGF, and the amino acid sequence ofCTGF, have been published. '040 Patent, SEQ ID NO:7 and SEQ ID NO:8,respectively.

Another apparently related protein is the mouse protein Fisp12(FIbroblast Secreted Protein). Fisp12 has been subjected to amino acidsequence analysis, revealing a primary structure that is rich incysteines. Ryseck et al., Cell Growth & Diff 2:225-233 (1991),incorporated herein by reference. The protein also possesses ahydrophobic N-terminal sequence suggestive of the signal sequencecharacteristic of secreted proteins.

Sequence analyses involving Cyr61, Fisp12, CTGF, and other proteins,have contributed to the identification of a family of cysteine-richsecreted proteins. Members of the family share similar primarystructures encoded by genes exhibiting similar sequences. Each of theproteins in this emerging family is further characterized by thepresence of a hydrophobic N-terminal signal sequence and 38 cysteineresidues in the secreted forms of the proteins. Members of the familyidentified to date include the aforementioned Cyr61 (human and mouse),Fisp12 (mouse), and CTGF (the human ortholog of Fisp12), as well asCEF10 (chicken), and Nov (avian).

One of several applications for a purified protein able to affect celladhesion, migration, and proliferation properties involves thedevelopment of stable, long term ex vivo hematopoietic stem cellcultures. Patients subjected to high-dose chemotherapy have suppressedhematopoiesis; expansion of stem cells, their maturation into varioushematopoietic lineages, and mobilization of mature cells intocirculating blood routinely take many weeks to complete. For suchpatients, and others who need hematopoietic cell transplantation,introduction into those patients of autologous stem cells that have beenmanipulated and expanded in culture is advantageous. Such hematopoicticstem cells (HSC) express the CD34 stem cell antigen, but do not expresslineage commitment antigens. These cells can eventually give rise to allblood cell lineages (e.g., erythrocytes, lymphocytes, and myelocytes).Hematopoietic progenitor cells that can initiate and sustain long termcultures (i.e., long term culture system-initiating cells or LTC-IC)represent a primitive population of stem cells. The frequency of LTC-IChas been estimated at only 1-2 per 10⁴ cells in normal human marrow andonly about 1 per 50-100 cells in a highly purified CD34⁺ subpopulation.Thus, it would be useful to have methods and systems for long term cellculture that maintain and expand primitive, pluripotent human HSC to beused for repopulation of the hematopoietic system in vivo.

Cell culture models of hematopoiesis have revealed a multitude ofcytokines that appear to play a role in the hematopoictic process,including various colony stimulating factors, interleukins, stem cellfactor, and the c-kit ligand. However, in ex vivo cultures, differentcombinations of these cytokines favor expansion ofdifferent setsofcommitted progenitors. For example, a factor in cord blood plasmaenhanced expansion of granulocyte-erythroid-macrophage-megakaryocytecolony forming unit (CFU-GEMM) progenitors, but expansion in thesecultures favored the more mature subsets of cells. Therefore, it hasbeen difficult to establish a culture system that mimics in vivohematopoiesis.

An HSC culture system should maintain and expand a large number ofmulti- or pluripotent stem cells capable of both long term repopulationand eventual lineage commitment under appropriate induction. However, inmost ex vivo culture systems, the fraction of the cell populationcomprised of LTC-IC decreases steadily with continued culturing, oftendeclining to 20% of their initial level after several weeks, as theculture becomes populated by more mature subsets of hematopoieticprogenitor cells that are no longer pluripotent. Moreover, theproliferative capacity exhibited by individual LTC-IC may varyextensively. Thus, a need exists in the art for HSC culture systemscomprising biological agents that maintain or promote the pluripotentpotential of cells such as LTC-IC cells. In addition to a role indeveloping ex vivo HSC cultures, biological agents affecting celladhesion, migration, and proliferation are useful in a variety of othercontexts.

Proteins that potentiate the activity of mitogens but have no mitogenicactivity themselves may play important roles as signaling molecules insuch processes as hematopoiesis. Moreover, these signaling proteinscould also serve as probes in the search for additional mitogens, manyof which have not been identified or characterized. Several biologicalfactors have been shown to potentiate the mitogenic activity of otherfactors, without being mitogenic themselves. Some of these potentiatorsare associated with the cell surface and/or extracellular matrix.Included in this group are a secreted basic Fibroblast GrowthFactor-binding protein (bFGF-binding protein), the basal lamina proteinperlecan, and the Human Immunodeficiency Viris-1 TAT protein, eachprotein being able to promote bFGF-induced cell proliferation andangiogenesis. Also included in this group of mitogen potentiators arethrombospondin, capable of activating a latent form of TransformingGrowth Factor-β, and an unidentified secreted growth-potentiating factorfrom vascular smooth muscle cells (Nakano et al. J. Biol. Chem.270:5702-5705 [1995]), the latter factor being required for efficientactivation of Epidermal Growth Factor- or thrombin-induced DNAsynthesis. Further, the B cell stimulatory factor-1/interleukin-4, a Tcell product with no demonstrable mitogenic activity, is able to 1)enhance the proliferative response of granulocyte-macrophage progenitorsto granulocyte-colony stimulating factor, 2) enhance the proliferativeresponse of erythroid progenitors to erythropoietin, and 3) togetherwith erythropoietin, induce colony formation by multipotent progenitorcells. Similarly, interleukin-7 enhanced stem cell factor-induced colonyformation by primitive murine bone marrow progenitors, althoughinterleukin-7 had no proliferative effect by itself. In addition,lymphocyte growth enhancing factor (LGEF) was found to enhancemitogen-stimulated human peripheral blood lymphocyte (PBL) or purified Tcell proliferation in a dose-dependenit fashion. LGEF alone did notstimulate PBL or T cell proliferation.

Therefore, a need continues to exist for biological agents capable ofexerting a concerted and coordinated influence on one or more of theparticularized functions (e.g., cell adhesion, cell migration and cellproliferation) collectively characterizing such complex biologicalprocesses as angiogenesis, chondrogenesis, and oncogenesis. In addition,a need persists in the art for agents contributing to the reproductionof these in vivo processes in an ex vivo environment, e.g., thedevelopment of HSC cultures. Further, there continues to be a need fortools to search for the remaining biological components of these complexprocesses, e.g., mitogen probes, the absence of which impedes efforts toadvantageously modulate and thereby control such processes.

SUMMARY OF THE INVENTION

The present invention provides extracellular matrix (ECM) signalingmolecule-related materials and methods. In particular, the presentinvention is directed to polynucleotides encoding ECM signalingmolecules and fragments or analogs thereof, ECM signalingmolecule-related polypeptides and fragments, analogs, and derivativesthereof, methods of producing ECM signaling molecules, methods of usingECM signaling molecules, methods of screening for modulators of an ECMsignaling molecule activity, those modulators, and methods of usingthose modulators to treat diseases or conditions related toangiogenesis, chondrogenesis, oncogenesis, cell migration, cell adhesionand cell proliferation.

One aspect of the present invention relates to a purified and isolatedpolypeptide comprising an ECM signaling molecule. The polypeptidesaccording to the invention retain at least one biological activity of anECM signaling molecule, such as the ability to stimulate cell adhesion,cell migration, or cell proliferation; the ability to modulateangiogenesis, chondrogenesis, or oncogenesis; immunogenicity or theability to elicit an immune response; and the ability to bind topolypeptides having specific binding sites for ECM signaling molecules,including antibodies and integrins. The polypeptides may be native orrecombinant molecules. Further, the invention comprehends full-lengthECM signaling molecules, and fragments thereof, such as an isolatedhuman Cyr61 fragment comprising a sequence selected from the groupconsisting of residues 280-290 of SEQ ID NO:33 and residues 305-310 ofSEQ ID NO:33, wherein said Cyr61 fragment retains at least onebiological function of human Cyr61. Of course, the human Cyr61 fragmentmay comprise the sequence set forth in SEQ ID NO:33, provided such afragment, like all of the fragments of the invention, retains at leastone biological function of human Cyr61. More generally, the human Cyr61fragment may comprise a sequence that is at least 95% similar to thesequence set forth in SEQ ID NO:33, wherein the human Cyr61 fragmentagain retains at least one biological function of human Cyr61. HumanCyr61 fragments contemplated by the invention may be encoded by apolynucleotide comprising a sequence that is at least 95% similar (usingBLAST software as described with default settings) to a polynucleotideencoding a polypeptide having the sequence set forth at SEQ ID NO:33,wherein said polypeptide retains at least one biological function ofhuman Cyr61. In addition, the polypeptides of the invention may beunderivatized, or derivatized in conformity with a native or non-nativederivatization pattern. The invention further extends to polypeptideshaving a native or naturally occurring amino acid sequence, and variants(i.e., polypeptides having different amino acid sequences), analogs(i.e., polypeptides having a non-standard amino acid or other structuralvariation from the conventional set of amino acids) and homologs (i.e.,polypeptides sharing a common evolutionary ancestor with anotherpolypeptide) thereof. Polypeptides that are covalently linked to othercompounds, such as polyethylene glycol, or other proteins or peptides,i.e., fusion proteins, are contemplated by the invention.

Exemplary ECM signaling molecules include mammalian Cyr61, Fisp12, andCTGF polypeptides. Beyond ECM signaling molecules, the inventionincludes polypeptides that specifically bind an ECM signaling moleculeof the invention, such as the aforementioned antibody products. A widevariety of antibody products fall within the scope of the invention,including polyclonal and monoclonal antibodies, antibody fragments,chimeric antibodies, CDR-grafted antibodies, “humanized” antibodies, andother antibody forms known in the art. Other molecules such as peptides,carbohydrates or lipids designed to bind to an active site of the ECMmolecules thereby inhibiting their activities are also contemplated bythe invention. However molecules such as peptides that enhance orpotentiate the activities of ECM molecule are also within the scope ofthe invention. The invention further extends to a pharmaceuticalcomposition comprising a biologically effective amount of a polypeptideand a pharmaceutically acceptable adjuvant, diluent or carrier,according to the invention. A “biologically effective amount” of thebiomaterial is an amount that is sufficient to result in a detectableresponse in the biological sample when compared to a control lacking thebiomaterial.

Another aspect of the invention relates to a purified and isolatedpolynucleotide comprising a sequence that encodes a polypeptide of theinvention. A polynucleotide according to the invention may be DNA orRNA, single- or double-stranded, and may be may purified and isolatedfrom a native source, or produced using synthetic or recombinanttechniques known in the art. The invention also extends topolynucleotides encoding fragments, analogs (i.e., polynucleotideshaving a non-standard nucleotide), homologs (i.e., polynucleotideshaving a common evolutionary ancestor with another polynucleotide),variants (i.e., polynucleotides differing in nucleotide sequence), andderivatives (i.e., polynucleotides differing in a structural manner thatdoes not involve the primary nucleotide sequence) of ECM molecules.Vectors comprising a polynucleotide according to the invention are alsocontemplated. In addition, the invention comprehends host cellstransformned or transfected with a polynucleotide or vector of theinvention.

In a related aspect, the invention contemplates a mammalian cellcomprising a cyr61 mutation selected from the group consisting of aninsertional inactivation of a cyr61 allele and a deletion of a portionof a cyr61 allele. The mammalian cell is preferably a human cell and themutation is either heterozygous or homozygous. The mutation, resultingfrom insertional inactivation or deletion, is either in the codingregion or a flanking region essential for expression such as a 5′promoter region. Cells are also found associated with non-human animals.

Other aspects of the invention relate to methods for making or using thepolypeptides and/or polynucleotides of the invention. A method formaking a polypeptide according to the invention comprises expressing apolynucleotide encoding a polypeptide according to the present inventionin a suitable host cell and purifying the polypeptide. Other methods formaking a polypeptide of the invention use techniques that are known inthe art, such as the isolation and purification of native polypeptidesor the use of synthetic techniques for polypeptide production. Inparticular, a method of purifying an ECM signaling molecule such ashuman Cyr61 comprises the steps of identifying a source containing humanCyr61, exposing the source to a human Cyr61-specific biomolecule thatbinds Cyr61 such as an anti-human Cyr61 antibody, and eluting the humanCyr61 from the antibody or other biomolecule, thereby purifying thehuman Cyr61.

Another aspect of the invention is a method of screening for a modulatorof angiogenesis comprising the steps of: (a) contacting a firstbiological sample capable of undergoing angiogenesis with a biologicallyeffective (i.e., angiogenically effective) amount of an ECM signalingmolecule-related biomaterial and a suspected modulator (inhibitor orpotentiator); (b) separately contacting a second biological sample witha biologically effective amount of an ECM signaling molecule-relatedbiomaterial, thereby providing a control; (c) measuring the level ofangiogenesis resulting from step (a) and from step (b); and (d)comparing the levels of angiogenesis measured in step (c), whereby amodulator of angiogenesis is identified by its ability to alter thelevel of angiogenesis when compared to the control of step (b). Themodulator may be either a potentiator or inhibitor of angiogenesis andthe ECM signaling molecule-related biomaterial includes, but is notlimited to, Cyr61, and fragments, variants, homologs, analogs,derivatives, and antibodies thereof.

The invention also extends to a method of screening for a modulator ofangiogenesis comprising the steps of: (a) preparing a first implantcomprising Cyr61 and a second implant comprising Cyr61 and a suspectedmodulator of Cyr61 angiogenesis; (b) implantling the first implant in afirst cornea of a test animal and the second implant in a second corneaof the test animal; (c) measuring the development of blood vessels inthe first and second corneas; and (d) comparing the levels of bloodvessel development measured in step (c), whereby a modulator ofangiogenesis is identified by its ability to alter the level of bloodvessel development in the first cornea when compared to the blood vesseldevelopment in the second cornea.

Another aspect of the invention is a method of screening for a modulatorof angiogeniesis comprising the steps of: (a) contacting a firstendothelial cell comprising a cyr61 allele with a suspected modulator ofangiogenesis; (b) measuring the Cyr61 activity of the first endothelialcell; (c) measuring the Cyr61 activity of a second endothelial cellcomprising a cyr61 allele; and (d) comparing the levels of Cyr61activity measured in steps (b) and (c), thereby identifying a modulatorof angiogenesis. A related aspect of the invention is drawn to a methodof screening for a modulator of angiogenesis comprising the steps of:(a) contacting a first endothelial cell with a polypeptide selected fromthe group consisting of a Cyr61, a Fisp12, a CTGF, a NOV, an ELM-1(WISP-1), a WISP-3, a COP-1 (WISP-2), and fragments, analogs, andderivatives of any of the aforementioned polypeptides, which are membersof the CCN family of proteins; (b) further contacting the firstendothelial cell with a suspected modulator of angiogenesis; (c)contacting a second endothelial cell with the polypeptide of step (a);(d) measuring the angiogenesis of the first endothelial cell; (c)measuring the angiogenesis of the second endothelial cell; and (f)comparing the levels of angiogenesis measured in steps (d) and (e),thereby identifying a modulator of angiogenesis.

Yet another related aspect of the invention is a method of screening formodulators of angiogenesis comprising the steps of: (a) constructing atransgenic animal comprising a mutant allele of a gene encoding apolypeptide selected from the group consisting of a Cyr61, a Fisp12, aCTGF, a NOV, an ELM-1 (WISP-1), a WISP-3, a COP-1 (WISP-2); (b)contacting the transgenic animal with a suspected modulator ofangiogenesis; (c) further contacting a wild-type animal with thepolypeptide, thereby providing a control; (d) measuring the levels ofangiogenesis in the transgenic animal; (e) measuring the level ofangiogenesis of the wild-type animal; and (f) comparing the levels ofangiogeniesis measured in steps (d) and (e), thereby identifying amodulator of angiogenesis.

Another aspect of the invention relates to a method of screening for amodulator of chondrogenesis comprising the steps of: (a) contacting afirst biological sample capable of undergoing chondrogeniesis with abiologically effective (e.g. chondrogenically effective) amount of anECM signaling molecule-related biomaterial and a suspected modulator;(b) separately contacting a second biological sample capable ofundergoing chondrogenesis with a biologically effective amount of an ECMsignaling molecule-related biomaterial, thereby providing a control; (c)measuring the level of chondrogenesis resulting from step (a) and fromstep (b); and (d) comparing the levels of chondrogenesis measured instep (c), whereby a modulator of chondrogenesis is identified by itsability to alter the level of chondrogenesis when compared to thecontrol of step (b). The modulator may be either a promoter or aninhibitor of chondrogenesis; the ECM signaling molecules include thosedefined above and compounds such as mannose-6-phosphate, heparin, andtenascin.

The invention also relates to an in vitro method of screening for amodulator of oncogenesis comprising the steps of: (a) inducing a firsttumor and a second tumor; (b) administering a biologically effectiveamount of an ECM signaling molecule-related biomaterial and a suspectedmodulator to the first tumor; (c) separately administering abiologically effective amount of an ECM signaling molecule-relatedbiomaterial to the second tumor, thereby providing a control; (d)measuring the level of oncogenesis resulting from step (b) and from step(c); and (e) comparing the levels of oncogenesis measured in step (d),whereby a modulator of oncogenesis is identified by its ability to alterthe level of oncogenesis when compared to the control of step (c).Modulators of oncogenesis contemplated by the invention includeinhibitors of oncogenesis. Tumors may be induced by a variety oftechniques including, but not limited to, the administration ofchemicals, e.g., carcinogens, and the implantation of cancer cells. Arelated aspect of the invention is a method for treating a solid tumorcomprising the step of delivering a therapeutically effective amount ofa Cyr61 inhibitor to an individual, thereby inhibiting theneovascularization of the tumor. Inhibitors include, but are not limitedto, inhibitor peptides such as peptides having the “RGD” motif, andcytotoxins, which may be free or attached to molecules such as Cyr61.

Yet another aspect of the invention is directed to a method of screeningfor a modulator of cell adhesion comprising the steps of: (a) preparinga surface compatible with cell adherence; (b) separately placing firstand second biological samples capable of undergoing cell adhesion on thesurface; (c) contacting a first biological sample with a suspectedmodulator and a biologically effective amount of an ECM signalingmolecule-related biomaterial selected from the group consisting of ahuman Cyr61, a human Cyr61 fragment, a human Cyr61 analog, and a humanCyr61 derivative; (d) separately contacting a second biological samplewith a biologically effective amount of an ECM signalingmolecule-related biomaterial selected from the group consisting of ahuman Cyr61, a human Cyr61 fragment, a human Cyr61 analog, and a humanCyr61 derivative, thereby providing a control; (c) measuring the levelof cell adhesion resulting from step (c) and from step (d); and (f)comparing the levels of cell adhesion measured in step (e), whereby amodulator of cell adhesion is identified by its ability to alter thelevel of cell adhesion when compared to the control of step (d).

In a related aspect, the invention provides a method of screening for amodulator of cell adhesion comprising the steps of: (a) contacting afirst fibroblast cell with a suspected modulator of cell adhesion and abiologically effective amount of an ECM signaling molecule-relatedbiomaterial selected from the group consisting of a Cyr61, a Fisp12, aCTGF, a NOV, an ELM-1 (WISP-1), a WISP-3, a COP-1 (WISP-2), andfragments, analogs, and derivatives of any of the aforementioned membersof the CCN family of proteins; (b) separately contacting a secondfibroblast cell with a biologically effective amount of an ECM signalingmolecule-related biomaterial described above, thereby providing acontrol; (c) measuring the level of cell adhesion resulting from step(a) and from step (b); and (d) comparing the levels of cell adhesionmeasured in step (c), whereby a modulator of cell adhesion is identifiedby its ability to alter the level of cell adhesion when compared to thecontrol of step (h). In a preferred embodiment of the method, thefibroblast cells present the α₆β₁ integrin. Also preferred arefibroblast cells that present a sulfated proteoglycan, such as a heparansulfate proteoglycan or a chondroitin sulfate proteoglycan.

Yet another aspect of the invention is a method of screening formodulators of macrophage adhesion comprising the steps of: (a)contacting a first macrophage with a polypeptide of the CCN family, suchas Cyr61, and a suspected modulator; (b) further contacting a secondmacrophage with the polypeptide of step (a); (c) measuring the bindingof the first macrophage to the polypeptide; (d) measuring the binding ofthe second macrophage to the polypeptide; and (e) comparing the bindingmeasurements of steps (d) and (e), thereby identifying a modulator ofmacrophage adhesion. Analogous methods of the invention are used toscreen for modulators of an inflammatory response.

The invention also extends to a method of screening for a modulator ofcell migration comprising the steps of: (a) forming a gel matrixcomprising Cyr61 and a suspected modulator of cell migration; (b)preparing a control gel matrix comprising Cyr61; (c) seeding endothelialcells capable of undergoing cell migration onto the gel matrix of step(a) and the control gel matrix of step (b); (d) incubating theendothelial cells; (e) measuring the levels of cell migration byinspecting the interior of the gel matrix and the control gel matrix forcells; (f) comparing the levels of cell migration measured in step (e),whereby a modulator of cell migration is identified by its ability toalter the level of cell migration in the gel matrix when compared to thelevel of cell migration in the control gel matrix. The endothelial cellsinclude, but are not limited to, human cells, e.g., human microvascularendothelial cells. The matrix may be formed from gelling materials suchas Matrigel, collagen, or fibrin, or combinations thereof.

In a related aspect, the invention comprehends a method of screening formodulators of fibroblast cell migration comprising the steps of: (a)contacting a first fibroblast cell with a suspected modulator of cellmigration and a biologically effective amount of an ECM signalingmolecule-related biomaterial selected from the group consisting of aCyr61, a Fisp12, a CTGF, a NOV, an ELM-1 (WISP-1), a WISP-3, a COP-1(WISP-2), and fragments, analogs, and derivatives of any of theaforementioned members of the CCN family of proteins; (b) separatelycontacting a second fibroblast cell with a biologically effective amountof an ECM signaling molecule-related biomaterial described above,thereby providing a control; (c) measuring the level of cell migrationresulting from step (a) and from step (b); and (d) comparing the levelsof cell migration measured in step (c), whereby a modulator of cellmigration is identified by its ability to alter the level of cellmigration when compared to the control of step (b). Preferredembodiments of the methods of screening for modulators of cell migrationinvolve the use of fibroblasts presenting an α₆β₁ integrin and/or asulfated proteoglycan.

Another aspect of the invention is directed to an in vitro method ofscreening for cell migration comprising the steps of: (a) forming afirst gelatinized filter and a second gelatinized filter, each filterhaving two sides; (b) contacting a first side of each the filter withendothelial cells, thereby adhering the cells to each the filter; (c)applying an ECM signaling molecule and a suspected modulator of cellmigration to a second side of the first gelatinized filter and an ECMsignaling molecule to a second side of the second gelatinized filter;(d) incubating each the filter; (e) detecting cells on the second sideof each the filter; and (f) comparing the presence of cells on thesecond side of the first gelatinized filter with the presence of cellson the second side of the second gelatinized filter, whereby a modulatorof cell migration is identified by its ability to alter the level ofcell migration measured on the first gelatinized filter when compared tothe cell migration measured on the second gelatinized filter. Theendothelial cells are defined above. The ECM signaling molecules extendto human Cyr61 and each of the filters may be placed in apparatus suchas a Boyden chamber, including modified Boyden chambers.

The invention also embraces an in vivo method of screening for amodulator of cell migration comprising the steps of: (a) removing afirst central portion of a first biocompatible sponge and a secondcentral portion of a second biocompatible sponge; (b) applying an ECMsignaling molecule and a suspected modulator to the first centralportion and an ECM signaling molecule to the second central portion; (c)reassociating the first central portion with said first biocompatiblesponge and said second central portion with the second biocompatiblesponge; (d) attaching a first filter to a first side of the firstbiocompatible sponge and a second filter to a second side of the firstbiocompatible sponge; (e) attaching a third filter to a first side ofthe second biocompatible sponge and a fourth filter to a second side ofthe second biocompatible sponge; (f) implanting each of thebiocompatible sponges, each biocompatible sponge comprising the centralportion and the filters, in a test animal; (e) removing each the spongefollowing a period of incubation; (f) measuring the cells found withineach of the biocompatible sponges; and (g) comparing the presence ofcells in the first biocompatible sponge with the presence of cells inthe second biocompatible sponge, whereby a modulator of cell migrationis identified by its ability to alter the level of cell migrationmeasured using the first biocompatible sponge when compared to the cellmigration measured using the second biocompatible sponge. ECM signalingmolecules include, but are not limited to, human Cyr61; the ECMsignaling molecule may also be associated with Hydron. In addition, thein vivo method of screening for a modulator of cell migration mayinclude the step of providing a radiolabel to the test animal anddetecting the radiolabel in one or more of the sponges.

Another aspect of the invention relates to a method for modulatinghemostasis comprising the step of administering an ECM signalingmolecule in a phanmaceultically acceptable adjuvant, diluent or carrier.Also, the invention extends to a method of inducing wound healing in atissue comprising the step of contacting a wounded tissue with abiologically effective amount of an ECM signaling molecule, therebypromoting wound healing. The ECM signaling molecule may be provided inthe form of an ECM signaling molecule polypeptide or an ECM signalingmolecule nucleic acid, e.g., using a gene therapy technique. Forexample, the nucleic acid may comprise an expression control sequenceoperably linked to an ECM signaling molecule which is then introducedinto the cells of a wounded tissue. The expression of the codingsequence is controlled, e.g., by using a tissue-specific promoter suchas the K14 promoter operative in skin tissue to effect the controlledinduction of wound healing. The nucleic acid may include a vector suchas a Herpesvirus, an Adenovirus, an Adeno-associated Virus, aCytomegalovinis, a Baculovirus, a retrovirus, and a Vaccinia Virus.Suitable wounded tissues for treatment by this method include, but arenot limited to, skin tissue and lung epithelium.

A related method comprises administering a biologically effective amountof an ECM signaling molecule, e.g. Cyr61, to an animal to promote organregeneration. The impaired organ may be the result of trauma, e.g.surgery, or disease. Another method of the invention relates toimproving the vascularization of grafts, e.g., skin grafts. Anothermethod of the invention is directed to a process for promoting boneimplantation, including bone grafts. The method for promoting boneimplantation comprises the step of contacting a bone implant orreceptive site with a biologically effective (i.e., chondrogenicallyeffective) amount of an ECM signaling molecule. The contacting step maybe effected by applying the ECM signaling molecule to a biocompatiblewrap such as a biodegradable gauze and contacting the wrap with a boneimplant, thereby promoting bone implantation. The bone implants comprisenatural bones and fragments thereof, as well as inanimate natural andsynthetic materials that are biocompatible, such as prostheses. Inaddition to direct application of an ECM signaling molecule to a bone,prosthesis, or receptive site, the invention contemplates the use ofmatrix materials for controlled release of the ECM signaling molecule,in addition to such application materials as gauzes.

Still another related aspect of the invention is a method of screeningfor modulators of wound healing comprising the steps of: (a) contactinga first activated platelet with a polypeptide of the CCN family, such asCyr61, and a suspected modulator; (b) further contacting a secondactivated platelet with the polypeptide of step (a); (c) measuring thebinding of the first activated platelet to the polypeptide; (d)measuring the binding of the second activated platelet to thepolypeptide; and (e) comparing the binding measurements of steps (d) and(e), thereby identifying a modulator of wound healing. Preferably, thewound healing involves the participation of platelet binding in theprocess of blood clotting. Also preferred are platelets presenting theα_(11b)β₃ integrin.

Yet another aspect of the invention relates to a method of screening fora modulator of cell proliferation comprising the steps of: (a)contacting a first biological sample capable of undergoing cellproliferation with a suspected modulator and a biologically effective(i.e., mitogenically effective) amount of an ECM signalingmolecule-related biomaterial selected from the group consisting of ahuman Cyr61, a human Cyr61 fragment, a human Cyr61 analog, and a humanCyr61 derivative; (b) separately contacting a second biological samplecapable of undergoing cell proliferation with a biologically effectiveamount of an ECM signaling molecule-related biomaterial selected fromthe group consisting of a human Cyr61, a human Cyr61 fragment, a humanCyr61 analog, and a human Cyr61 derivative, thereby providing a control;(c) incubating the first and second biological samples; (d) measuringthe level of cell proliferation resulting from step (c); and (e)comparing the levels of cell proliferation measured in step (d), wherebya modulator of cell proliferation is identified by its ability to alterthe level of cell adhesion when compared to the control of step (b).

In a related aspect, the invention contemplates a method of screeningfor modulators of fibroblast cell proliferation comprising the steps of:(a) contacting a first fibroblast cell with a suspected modulator ofcell proliferation and a biologically effective amount of an ECMsignaling molecule-related biomaterial selected from the groupconsisting of a Cyr61, a Fisp12, a CTGF, a NOV, an ELM-1 (WISP-1), aWISP-3, a COP-1 (WISP-2), and fragments, analogs, and derivatives of anyof the aforementioned members of the CCN family of proteins; (b)separately contacting a second fibroblast cell with a biologicallyeffective amount of an ECM signaling molecule-related biomaterialdescribed above, thereby providing a control; (c) measuring the level ofcell proliferation resulting from step (a) and from step (b); and (d)comparing the levels of cell proliferation measured in step (c), wherebya modulator of cell proliferation is identified by its ability to alterthe level of cell proliferation when compared to the control of step(b). Preferred embodiments of the methods of screening for modulators ofcell proliferation involve the use of fibroblasts presenting an α₆β₁integrin and/or a sulfated proteoglycan.

A related aspect of the invention is a modulator of an ECM signalingmolecule identified using the screening methods of the invention. In oneembodiment, Cyr61 is the ECM signaling molecule whose activity ismodulated, induction of angiogenesis and/or cell adhesion is theactivity that is modulated, and the modulator is a peptide. A preferredpeptide modulator of Cyr61-induced angiogenesis and/or cell adhesion isa peptide having a sequence derived from domain III of Cyr61, includingpeptides having the sequence H 2N-GQKCIVQTTSWSQCSKS-CO₂H (SEQ ID NO: 33) and peptides having amino acid of 80%, 90%, 95% and preferably, 99%sequence similarity. The peptide modulators of the invention include ECMsignaling molecule fragments of varying length, variants thereofpreferably exhibiting conservative amino acid substitutions, additionsor deletions, and derivatives thereof, including, but not limited to,peptides that are glycosylated, PEGylated, phosphorylated, and attachedor fused to other peptides or non-peptide carrier molecules.

Also comprehended by the invention is a method for expanding apopulation of undifferentiated hematopoletic stem cells in culture,comprising the steps of: (a) obtaining hematopoietic stem cells from adonor; and (b) culturing said cells under suitable nutrient conditionsin the presence of a biologically effective (i.e., hematopoieticallyeffective) amount of a Cyr61 polypeptide.

Another method according to the invention is a method of screening for amitogen comprising the steps of: (a) plating cells capable of undergoingcell proliferation; (b) contacting a first portion of the cells with asolution comprising Cyr61 and a suspected mitogen; (c) contacting asecond portion of the cells with a solution comprising Cyr61, therebyproviding a control; (c) incubating the cells; (d) detecting the growthof the first portion of cells and the second portion of the cells; and(e) comparing growth of the first and second portions of cells, wherebya mitogen is identified by its ability to induce greater growth in thefirst portion of cells when compared to the growth of the second portionof cells. The cells include, but are not limited to, endothelial cellsand fibroblast cells. Further, the method may involve contacting thecells with a nucleic acid label, e.g., [³H]-thymidine, and detecting thepresence of the label in the cells. Another method relates to improvingtissue grafting, comprising administering to an animal a quantity ofCyr61 effective in improving the rate of neovascularization of a graft.

In other aspects, the invention is drawn to a method of screening for amodulator of binding between a Cyr61 polypeptide and a Cyr61 receptorintegrin selected from the group consisting of α_(V)β₃, α_(V)β₅, α₆β₁,α₁₁β₃ and α_(M)β₂, comprising the steps of: a) contacting a Cyr61polypeptide with an integrin receptor composition in the presence and inthe absence of a potential modulator compound; b) detecting bindingbetween the polypeptide and the integrin receptor; and c) identifying amodulator compound in view of decreased or increased binding between theCyr61 polypeptide and the integrin receptor in the presence of themodulator, as compared to binding in the absence of the modulator.Suitable Cyr61 polypeptides include human Cyr61, and fragments, analogsand derivative thereof. For example, a preferred Cyr61 fragment wouldhave at least 95% amino acid similarity to residues 1-281 of SEQ IDNO:4, thereby containing domains I-III, but not domain IV, of Cyr61.Purified modulators identified by the above-described method ofscreening for a modulator of binding between a Cyr61 polypeptide and aparticular integrin receptor are also embraced by the invention.Further, the invention contemplates the use of a modulator of aCyr61-integrin α_(V)β₃ interaction for preparation of a medicament forthe treatment of a condition selected from the group consisting ofatherosclerosis, heart disease, tumor growth, tumor metastasis,fibrosis, disorders associated with inadequate angiogenesis, disordersassociated with aberrant granulation tissue development, aberrantfibroblast growth and wounds.

In still another aspect, the invention provides a method for treating acondition characterized by defective connective tissue in a mammaliansubject, comprising the steps of: (a) identifying a mammalian subject inneed of treatment for the condition, and (b) administering to themammalian subject a composition comprising a modulator of an interactionbetween Cyr61 and an integrin receptor selected from the groupconsisting of α_(V)β₃, α_(V)β₅, α₆β₁, α₁₁β₃ and α_(Mβ) ₂, in an amounteffective to mitigate the symptoms of the condition in the mammaliansubject. Suitable modulators include, but are not limited to, amodulator selected from the group consisting of heparin, heparan sulfateand a polypeptide that binds an integrin receptor. The modulator may beadministered in a wide variety of ways, including the situation whereadministering is accomplished by expressing an exogenous polypeptidecoding region in cells of the type affected by said condition. Anexemplary polypeptide is a fragment of a Cyr61 polypeptide, for examplea fragment comprising a sequence selected from the group consisting ofresidues 280-290 of SEQ ID NO:4 and residues 306-312 of SEQ ID NO:4.Further, a variety of conditions or disorders (e.g., diseases) andintegrin receptors are contemplated, such as a condition that involves adefect in fibroblast adhesion and the integrin receptor is α₆β₁, acondition that involves a defect in fibroblast chemotaxis and theintegrin receptor is α_(V)β₅, and a condition that involves a defect infibroblast proliferation and the integrin receptor is α_(V)β₃.

Another aspect of the invention is drawn to methods of treatingconditions or disorders, such as diseases, associated with gene under-or over-expression by delivering a therapeutically effective amount ofan ECM Signaling Molecule (e.g., a Cyr61 polypeptide, Fisp12, CTGF), ora biologically active fragment thereof, or modulator of a Cyr61-integrinreceptor interaction, using delivery means known in the art. A relatedaspect of the invention is drawn to a method for modulating geneexpression comprising the step of administening a biologically effectiveamount of a human Cyr61 fragment to a cell capable of Cyr61 -modulatedgene expression.

In yet another aspect, the invention is drawn to a method for treating acondition characterized by a defect in smooth muscle tissue in amammalian subject, comprising the steps of: (a) identifying a mammaliansubject in need of treatment for the condition, and (b) administering tothe mammalian subject a composition comprising a modulator of aninteraction between Cyr61 and an α₆β₁ integrin receptor in an amounteffective to mitigate the symptoms of the condition in the mammaliansubject. An exemplary modulator is selected from the group consisting ofheparin, heparan sulfate and a polypeptide that binds an α₆β₁ integrinreceptor. One of many suitable means of administering the composition isaccomplished by expressing an exogenous polypeptide coding region incells of the type affected by said condition. Again, exemplary Cyr61polypeptides include fragments of Cyr61, such as a fragment thatcomprises at sequence selected from the group consisting of residues280-290 of SEQ ID NO:4 and residues 306-312 of SEQ ID NO:4. Conditionsthat may be treated by the method include a condition selected from thegroup consisting of atherosclerosis, heart disease, tumor growth, tumormetastasis, fibrosis, disorders associated with inadequate angiogenesis,disorders associated with aberrant granulation tissue development,aberrant fibroblast growth and wounds.

Another aspect of the invention is directed to a kit for assaying forCyr61-integrin receptor interactions, the kit comprising a Cyr61polypeptide and a composition comprising an integrin receptor selectedfrom the group consisting of α_(V)β₃, α_(V)β₅, α₆β₁, α₁₁β₃ and α_(M)β₂.An exemplary kit contains an integrin receptor composition thatcomprises an α_(V)β₃ integrin localized in a mammalian fibroblast cellmembrane.

Numerous additional aspects and advantages of the present invention willbe apparent upon consideration of the following drawing and detaileddescription.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 presents the comparative amino acid sequences of members of thecysteine-rich protein family of growth-regulating proteins.

DETAILED DESCRIPTION OF THE INVENTION

In the mouse, the Cyr61 protein has been found to influence celladhesion, migration, and proliferation. The cyr61 gene, which encodesCyr61, is an immediate-early gene that is transcriptionally activated byserum growth factors in mouse fibroblasts. Lau et al., EMBO J.4:3145-3151 (1985), incorporated herein by reference; Lau et al., Proc.Natl. Acad. Sci. (USA) 84:1182-1186 (1987), incorporated herein byreference. The murine cyr61 cDNA coding sequence is set forth in SEQ IDNO:1. (The human cyr61 cDNA coding sequence is provided in SEQ ID NO:3).The amino acid sequence of murine Cyr61 is set out in SEQ ID NO:2. (Thehuman Cyr61 amino acid sequence is presented in SEQ ID NO:4). Cyr61 is a41 kDa polypeptide exhibiting 39 cysteine residues, approximately 10% ofthe 379 amino acids constituting the unprocessed protein. Yang et al.,Cell Growth & Diff. 2:351-357 (1991), incorporated herein by reference.Investigations have revealed that murine Cyr61 binds heparin and issecreted. Yang et al. Consistent with the observed secretion of Cyr61 isthe identification of an N-terminal signal sequence in nascent Cyr61,deduced from inspection of the murine cyr61 cDNA sequence. Yang et al.Additionally, Cyr61 is not found in the conditioned medium of culturedcells expressing cyr61, but is found associated with the extracellularmatrix (ECM) and the cell surface. Yang et al. Structurally similarcysteine-rich mammalian proteins have been characterized.

Fisp12, a cysteine-rich murine protein, exhibits structural similarityto Cyr61. The cDNA sequence encoding Fisp12 is set forth in SEQ ID NO:5;the amino acid sequence of Fisp12 is presented in SEQ ID NO:6. MurineFisp12, like Cyr61, influences cell adhesion, proliferation andmigration. The human ortholog of Fisp12 is Connective Tissue GrowthFactor (CTGF), a protein similar in structure and function to Cyr61.Fisp 12, and CTGF, are distinguishable from Cyr61, however. For example,a greater proportion of secreted Fisp12 is found in the culture mediumthan is the case with Cyr61; a correspondingly lower proportion ofFisp12 is localized in the area of expressing cells (cell surface andnearby extracellular matrix) than is found with Cyr61. Additionalsimilarities and distinctions among the proteins comprising the ECMsignaling molecules of the invention will become apparent in therecitations below.

The present invention has multiple aspects, illustrated by the followingexamples. Example 1 describes the cloning of polynucleotides encodingmembers of the cysteine-rich protein family of ECM signaling molecules;Example 2 describes sequence analyses; Example 3 describes RNA analyses;Example 4 describes the production of transgenic animals; Example 5describes the expression of Cyr61 polypeptides; Example 6 describes theexpression of Fisp12 polypeptides; Example 7 sets out methods ofpolypeptide purification; Example 8 provides a characterization of thepolypeptides of the invention; Example 9 discloses a heparin bindingassay for the polypeptide members of the cysteine-rich protein family;Example 10 is directed to receptors for the polypeptides; Example 11describes anti-ECM signaling molecule antibodies; Example 12 is directedto inihibitory peptides; Example 13 describes cell adhesion andpolypeptide-based methods for influencing the process of cell adhesion,Example 14 describes polypeptide-influenced migration of fibroblasts;Example 15 describes the migration of endothelial cells and in vitroassays for migration; Example 16 describes an in vitro assay forinhibitors of endothelial cell migration; Example 17 describes an invivo assay for endothelial cell migration; Example 18 describes mitogenpotentiation by the polypeptides of the invention; Example 19 describesan in vivo cornea assay for angiogenic factors and modulators; Example20 is directed to methods for influencing blood clotting using thepolypeptides of the invention; Example 21 discloses the use of thepolypeptides for ex vivo hematopoietic stem cell cultures; Example 22addresses organ regeneration; Example 23 describes chondrogenesis andthe expression of extracellular matrix signaling molecules in mesenchymecells; Example 24 describes the promotion of cell adhesion in theprocess of chondrogenesis using the polypeptides of the invention;Example 25 describes chondrogenesis and the influence of thepolypeptides of the invention on cell aggregation; Example 26 describesthe promotion of cell proliferation by polypeptides of the invention inthe process of chondrogenesis; Example 27 addresses methods for usingthe polypeptides of the invention to affect chondrogenesis; Example 28provides genetic approaches to the use of polynucleotides of theinvention; Example 29 describes Fibroblast adhesion, Example 30addresses angiogenesis, Example 31 relates to insertional inactivationor knock-out genetic constructs, Example 32 describes adhesion toplatelets and macrophages, and Example 33 describes peptide modulatorsof Cyr61 activity. These examples are intended to be illustrative of thepresent invention and should not be construed to limit the scope of theinvention.

EXAMPLE 1 Polynucleotide Cloning

Initially, an attempt was made to isolate a human cyr61 cDNA from ahuman placental cDNA library by probing with the murine cyr61 cDNAsequence using techniques that are standard in the art. See Sambrook etal., incorporated herein by reference. Isolation of the complete murinecyr61 cDNA from a BALB/c 373 (ATCC CRL-1658) cDNA library has beendescribed. O'Brien et al., Mol. Cell. Biol. 10:3569-3577 (1990),incorporated herein by reference. The nucleotide and deduced amino acidsequences of murine cyr61 are available from the Genbank database underaccession number M32490. The nucleotide sequence of murine cyr61 ispresented in SEQ ID NO:1; the murine Cyr61 amino acid sequence ispresented in SEQ ID NO:2.

The human cDNA library was constructed using λgt11 (Promega Corp.,Madison, Wis.) as a vector which was transfected into E. coli and platedon LB agar. A murine cDNA expression construct cloned in pGEM-2 (O'Brienet al., [1990]), containing the entire murine cyr61 coding sequence[nucleotides 56-1560, using the numbering of O'Brien et al., (1990); seeSEQ ID NO:1] was used as a probe. The mouse cDNA probe was radiolabeledby techniques standard in the art. Sambrook et al. Plaque screeningsusing the mouse probe were performed using standard techniques. Sambrooket al.

More particularly, agar plates containing the human cDNA librarydescribed above were exposed to nitrocellulose filters (BA85, 82 mm,Schleicher & Schuell, Keene, N.H.) were placed on each plate. Afterplaque adsorption (approximately 20 minutes), the filters were removedand air dried for approximately 30 minutes. Subsequently, each filterwas sequentially submerged for 30-60 seconds in 0.2 M NaOH, 1.5 M NaCl(100 ml); 2× SSC, 0.4 M Tris-HCl, pH 7.4 (100 ml); and 0.2× SSC (100ml). Filters were then dried at room temperature for approximately 1hour and subjected to 80° C. under vacuum for 2 hours. Filters wereprobed with radiolabeled murine cyr61 cDNA. The excessive number ofsignals, indicative of a high level of false positive signals fromrelated sequences, prevented identification of cyr61 cDNA.

Turning to a complicated, cyr61-specific cloning strategy, human cyr61cDNA clones were identified with probes generated by RT-PCR. Inparticular, the probe for screening the human placental cDNA library wasa PCR fragment generated with degenerate primers by RT-PCR of total RNAfrom logarithmically growing WI38 cells. The primers were derived fromthe sequences corresponding to the most conserved region of the openreading frame of the mouse cyr61 cDNA. One primer, designated H61-5[5′-GGGAATTCTG(TC)GG(GATC)TG(TC)TG(TC)AA(GA)GT(GC)TG-3′ (SEQ ID NO: 39],contains a degenerate sequence which, with the exception of the“GGGAATTC” sequence at the 5′ end which was used to introduce an EcoRIsite, is derived from nuclcotides 327-346 (sense strand) of the mousecyr61 sequence set forth in ID NO:1. The degeneracies appear inpositions corresponding to the third position of codons in SEQ ID NO:1.The second primer used for PCR amplification of a human cyr61 sequencewas designated H61-3 [5′-CCGGATCC(GA)CA(GA)TT(GA)TA(GA)TT(GA)CA-3′ (SEQID NO: 40)]which, with the exception of the 5′ sequence “CCGGATCC” usedto introduce a BamHI site, corresponds to the anti-sense strandcomplementary to nucleotides 1236-1250of the mouse cyr61 sequence setforth in SEQ ID NO:1. The degeneracies occur in positions complementaryto the third positions of codons in mouse cyr61 as set forth in SEQ IDNO:1. The amplified cyr61 cDNA was cloned into the pBlueScript SK+vector (Stratagene, La Jolla, Calif.) and sequenced with a Sequenase IIkit (U.S. Biochemicals, Cleveland, Ohio).

Serial screenings of the human placental cDNA library led to theisolation of a clone containing a human cyr61 cDNA. The human cyr61 cDNAis approximately 1,500 bp in length. The human cDNA is contained on anEcoRI fragment cloned into the EcoRI site in pGEM-2. As shown in SEQ IDNO:3, the human cDNA sequence includes the entire coding region forhuman Cyr61, along with 120 bp of 5′ flanking sequence, and about 150 bpof 3′ flanking sequence.

The polynucleotides of the invention may be wholly or partiallysynthetic, DNA or RNA, and single- or double-stranded. Becausepolynucleotides of the invention encode ECM signaling moleculepolypeptides which may be fragments of an ECM signaling moleculeprotein, the polynucleotides may encode a partial sequence of an ECMsignaling molecule. Polynucleotide sequences of the invention are usefulfor the production of ECM signaling molecules by recombinant methods andas hybridization probes for polynucleotides encoding ECM signalingmolecules.

DNA polynucleotides according to the invention include genomic DNAs,cDNAs, and oligonucleotides comprising a coding sequence of an ECMsignaling molecule, or a fragment or analog of an ECM signalingmolecule, as described above, that retains at least one of thebiological activities of an ECM signaling molecule such as the abilityto promote cell adhesion, cell migration, or cell proliferation in suchbiological processes as angiogenesis, chondrogenesis, and oncogenesis,or the ability to elicit an antibody recognizing an ECM signalingmolecule.

Other polynucleotides according to the invention differ in sequence fromsequences contained within native ECM signaling molecule polynucleotides(i.e., by the addition, deletion, insertion, or substitution ofnucleotides) provided the polynucleotides encode a protein that retainsat least one of the biological activities of an ECM signaling molecule.A polynucleotide sequence of the invention may differ from a native ECMsignaling molecule polynucleotide sequence by silent mutations that donot alter the sequence of amino acids encoded therein. Additionally,polynucleotides of the invention may specify an ECM signaling moleculethat differs in amino acid sequence from native ECM signaling moleculesequences or subsequences, as described above. For example,polynucleotides encoding polypeptides that differ in amino acid sequencefrom native ECM signaling molecules by conservative replacement of oneor more amino acid residues, are contemplated by the invention. Theinvention also extends to polynucleotides that hybridize under standardstringent conditions to polynucleotides encoding an ECM signalingmolecule of the invention, or that would hybridize but for thedegeneracy of the genetic code. Exemplary stringent hybridizationconditions involve hybridization at 42° C. in 50% formamide, 5× SSC, 20mM Na.PO₄, pH 6.8 and washing in 0.2× SSC at 55° C. It is understood bythose of skill in the art that variation in these conditions occursbased on the length and GC nucleotide content of the sequences to behybridized. Formulas standard in the art are appropriate for determiningexact hybridization conditions. See Sambrook et al. Molecular Cloning: ALaboratory Manual (Second ed., Cold Spring Harbor Laboratory Press 1989)§§ 9.47-9.51.

ECM signaling molecule polynucleotides comprising RNA are also withinthe scope of the present invention. A preferred RNA polynucleotideaccording to the invention is an mRNA of human cyr61. Other RNApolynucleotides of the invention include RNAs that differ from a nativeECM signaling molecule mRNA by the insertion, deletion, addition, orsubstitution of nucleotides (see above), with the proviso that theyencode a polypeptide retaining a biological activity associated with anECM signaling molecule. Still other RNAs of the invention includeanti-sense RNAs (i.e., RNAs comprising an RNA sequence that iscomplementary to an ECM signaling molecule mRNA).

Accordingly, in another embodiment a set of DNA fragments collectivelyspanning the human cyr61 cDNA were cloned in pGEM-2 and M13 derivativesusing methods well known in the art to facilitate nucleotide sequenceanalyses. The pGEM-2 clones provided substrates for the enzymaticgeneration of serial deletions using techniques known in the art. Thiscollection of clones, collectively containing a series of DNA fragmentsspanning various parts of the cyr61 cDNA coding region, are useful inthe methods of the invention. The resulting series of nested pGEM-2clones, in turn, provided substrates for nucleotide sequence analysesusing the enzymatic chain terminating technique. The fragments are alsouseful as nucleic acid probes and for preparing Cyr61 deletion ortruncation analogs. For example, the cyr61 cDNA clones may be used toisolate cyr61 clones from human genomic libraries that are commerciallyavailable. (Clontech Laboratories, Inc., Palo Alto, Calif.). Genomicclones, in turn, may be used to map the cyr61 locus in the human genome,a locus that may be associated with a known disease locus.

Other embodiments involve the polynucleotides of the invention containedin a variety of vectors, including plasmid, viral (e.g., prokaryotic andeukaryotic viral vectors derived from Lambda phage, Herpesviruses,Adenovirus, Adeno-associated viruses, Cytomegalovirus, Vaccinia Virus,the M13-f1-fd family of viruses, retroviruses, Baculovirus, and others),phagemid, cosmid, and YAC (i.e., Yeast Artificial Chromosome) vectors.

Yet other embodiments involve the polynucleotides of the inventioncontained within heterologous polynucleotide environments.Polynucleotides of the invention have been inserted into heterologousgenomes, thereby creating transgenes, and transgenic animals, accordingto the invention. In particular, two types of gene fusions containingpartial murine cry61 gene sequences have been used to generatetransgenic mice. (See below). One tyne of fused gene recombined thecoding sequence of cyr61 with one of three different promoters: 1) theK14 keratin promoter, 2) the β-actin promoter, or 3) thephosphoglycerokinase promoter. Adra et al., Gene 60:65-74 (1987). Thesefusion constructs were generated using standard techniques, as describedbelow in the context of a phosphoglycerokinase promoter (pgk-1)-cyr61fusion. An XhoI-Sca1 genomic DNA fragment containing the entire cyr61coding region and all introns, but lacking the transcription initiationsite and polyadenylation signal, was cloned into plasmid pgk/β-gal,replacing the lacZ coding sequence. The resulting construct placed cyr61under the control of the strong pgk-1 promoter which is active in allcells.

The second type of gene fusion recombined the cyr61 expression controlsequences (i.e., promoter) with the E. coli β-galactosidase codingsequence. The cyr61-lacZ fusion gene was constructed using the followingapproach. A DNA fragment spanning nucleotides −2065 to +65 relative tothe transcription initiation nucleotide was used to replace the pgk-1promoter (Adra et al. [1987]) in plasmid pgk/β-gal by blunt-end cloning.In addition, the polyadenylation signal from the bovine growth honnonegene was cloned into the plasmid containing the fusion gene. Theresulting construct, plasmid 2/lacZ, has the E. coli lacZ gene under thetranscriptional control of a 2 kb DNA fragment containing the cyr61promoter. The related plasmid 1.4/lacZ was derived from plasmid 2lacZ byremoving about 600 bp of cyr61 DNA found upstream of an AflII site.Also, plasmid 2M/lacZ resembles plasmid 2/lacZ, except for a C-to-Ttransition in the CArG Box, created by PCR. These constructs wereexcised from the vectors by NotI digestion, purified using GeneClean(Bio101, Inc., La Jolla, Calif.), and used to generate transgenic mice(see below).

A cDNA fragment encoding mouse fisp12 has also been cloned usingstandard techniques. Ryseck et al., Cell Growth & Diff. 2:225-233(1991), incorporated herein by reference. The cloning was accomplishedby ligating an Xholl fragment containing the fisp12 cDNA coding regioninto BamHI-cleaved pBlueBacIII, a baculovirus expression vector(Invitrogen Corp., San Diego, Calif.). Recombinant baculovirus cloneswere obtained as described in Summers et al., TX Ag. Exp. Sta., Bulletin1555 (1987).

The human ortholog of fisp12, the gene encoding CTGF, was cloned byscreening a fusion cDNA library with anti-Platelet-Derived Growth Factor(anti-PDGF) antibodies, as described in U.S. Pat. No. 5,408,040, column12, line 16, to column 13, line 29, incorporated herein by reference.The screening strategy exploited the immunological cross-reactivity ofCTGF and PDGF.

The cloned copies of the cyr61, fisp12, and ctgf cDNAs provide a readysource for polynucleotide probes to facilitate the isolation of genomiccoding regions, as well as allelic variants of the genomic DNAs orcDNAs. In addition, the existing cDNA clones, or clones isolated byprobing as described above, may be used to generate transgenicorganisms. For example, transgenic mice harboring cyr61 have beengenerated using standard techniques, as described in the next Example.

A clone, hCyr61 cDNA, containing the human cyr61 cDNA sequence set forthin SEQ ID NO:3, and a bacterial strain transformed with that clone,Escherichia coli DH5α (hCyr61cDNA), were deposited with the AmericanType Culture Collection, 10801 University Blvd., Manassas, Va.20110-2209 USA (formerly at 12301 Parklawn Drive, Rockville, Md. 20852USA), on Mar. 14, 1997.

EXAMPLE 2 Sequence Analyses

The nucleotide sequence of murine cyr61 has been described, O'Brien etal. (1990); Latinkic et al. Nucl. Acids Res. 19:3261-3267 (1991), and isset out herein as SEQ ID NO:1.

The deduced amino acid sequence of murine Cyr61 has been reported,O'Brien et al. (1990), and is set forth in SEQ ID NO:2.

The nucleotide sequence of the human cyr61 cDNA was determined using themethod of Sanger, as described in Sambrook et al. Sequencing templateswere generated by constructing a series of nested deletions from apGEM-2 human cyr61 cDNA clone, as described in Example 1 above. Thehuman cyr61 cDNA sequence is set forth in SEQ ID NO:3. The amino acidsequence of human Cyr61 was deduced from the human cyr61 cDNA sequenceand is set forth in SEQ ID NO:4.

A comparison of the mouse and human Cyr61 sequences, presented in SEQ IDNO:2 and SEQ ID NO:4, respectively, reveals 91% similarity. Bothsequences exhibit an N-terminal signal sequence indicative of aprocessed and secreted protein; both proteins also contain 38 cysteineresidues, distributed throughout both proteins but notably absent fromthe central regions of both murine and human Cyr61. Notably, the regionof greatest sequence divergence between the mouse and human Cyr61 codingregions is this central region free of cysteine residues. However, the5′ untranslated regions of the mouse and human cyr61 cDNAs are even moredivergent (67% similarity). In contrast, the 3′ untranslated regions arethe most similar regions (91% similarity). In overall length, theencoded murine Cyr61 has 379 amino acids; human Cyr61 has 381 aminoacids.

A fisp12 cDNA sequence has also been determined and is set out in SEQ IDNO:5. The amino acid sequence of Fisp12 has been deduced from the fisp12cDNA sequence and is set forth in SEQ ID NO:6. A comparison of the aminoacid sequences of murine Cyr61 and Fisp12 reveals that the two proteinsare 65% identical. The structural similarity of Cyr61 and Fisp12 isconsistent with the similar functional properties of the two proteins,described below.

A partial cDNA sequence of CTGF, containing the complete CTGF codingregion, has also been determined. The CTGF cDNA sequence was obtainedusing M13 clones as templates for enzymatic sequencing reactions, asdescribed. '040 Patent, at column 12, line 68 to column 13, line 14.Additional cloning coupled with double-stranded enzymatic sequencingreactions, elucidated the entire sequence of the cDNA encoding CTGF.U.S. Pat. No. 5,408,040, column 14, line 44, to column 15, line 8,incorporated herein by reference. The nucleotide sequence of the cDNAencoding CTGF is presented herein in SEQ ID NO:7. The deduced amino acidsequence of the cDNA encoding CTGF is presented in SEQ ID NO:8.

EXAMPLE 3 RNA Analyses

Polynucleotide probes are useful diagnostic tools for angiogenic, andother, disorders correlated with Cyr61 expression because properlydesigned probes can reveal the location, and level, of cyr61 geneexpression at the transcriptional level. The expression of cyr61, inturn, indicates whether or not genes controlling the process ofangiogenesis are being expressed at typical, or expected, levels.

Using these tools, the mouse cyr61 mRNA expression pattern wasdetermined using an RNase protection technique. O'Brien et al., (1992).In particular, a 289 nucleotide antisense riboprobe was used that wouldprotect 246 nucleotides of the murine cyr61 mRNA (nucleotides 67 to 313using the numbering of O'Brien et al.) The assays showed levels of cyr61mRNA in PSA-1 cells (10 μg of total RNA) from either theundifferentiated state or stages 1, 2, and 3 of differentiation (PSA-1cells undergo three stages of cellular differentiation corresponding tomouse embryonic cells of the following gestational ages, in days:4.5-6.5 [PSA-1 stage 1]; 6.5-8.5 [PSA-1 stage 2]; 8.5-10.5 [PSA-1 stage3]). A comparison of the protection of whole embryonic and placentaltotal RNAs (20 μg each) showed that cyr61 is expressed in embryonictissues at times that are coincident with the processes of celldifferentiation and proliferation.

Expression characteristics of human cyr61 were determined by Northernanalyses, using techniques that are standard in the art. Sambrook et al.RNA was isolated from the human diploid fibroblastic cell line WI38(ATCC CCL-75). In addition, RNA was isolated from rat cells (REF52),hamster cells (CHO), and monkey cells (BSC40). Each of the cell lineswas grown to confluence in MEM-10 (Eagle's Minimal Essential Medium withEarle's salts [GIBCO-BRL, Inc.], 2 mM glutamine, and 10% fetal calfserum [fcs]) and maintained in MEM-0.5 (a 0.5% serum medium) for twodays. Cultures were then stimulated with 20% fcs, in the presence orabsence of cycloheximide, by techniques known in the art. Lau et al.(1985; 1987). Ten microgram aliquots of RNA isolated from these celllines were then fractionated by formaldehyde-agarose gelelectrophoresis, transferred and immobilized on nitrocellulose filters,and exposed to a full-length [³²P]-radiolabeled murine cyr61 cDNA probeunder hybridization conditions of high stringency. Human cyr61 RNAexpression was similar to murine cyr61 expression. Both mouse and humancyr61 expression yielded approximately 2 kilobase RNAs. Additionally,both mouse and human expression of Cyr61 were stimulated by serum andwere resistant to cycloheximide.

The distribution of human cyr61 mRNA was also examined using multipletissue Northem blots (Clontech). The blots were hybridized in anExpressHyb Solution (Clontech) according to the manufacturer'sinstructions. The results showed that cyr61 mRNA is abundant in thehuman heart, lung, pancreas, and placenta; is present at low levels inskeletal muscle, kidney and brain; and is not detectable in liver. Theseresults are consistent with the expression of cyr61 in mouse tissues.

In addition, total cellular RNA was isolated from human skin fibroblasts(HSFs) that were either quiescent, growing exponentially, stimulated byserum, or exposed to cycloheximide. HUVE cells (ATCC CRL 1730) weremaintained in Ham's F12 medium supplemented with 10% fbs (Intergene),100 μg/ml heparin (Gibco BRL) and 30 μg/ml endothelial cell growthsupplement (Collaborative Biomedical Products). Human skin fibroblasts(HSF, ATCC CRL-1475) and WI38 fibroblasts (ATCC CCL-75) were grown inDulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fbs.Quiescent HSFs were prepared by growth in DMEM supplemented with 10% fbsto confluence followed by changing the medium to DMEM containing 0.1%fbs, for 2 days. Serum stimulation was carried out by changing themedium to 20% fbs for 1 hour.

Where indicated, cycloheximide was added to 10 μg/ml simultaneously withserum for 3 hours.

RNAs from the aforementioned cells were isolated using a guanidiniumisothiocyanate protocol. Chomczynski et al., Anal. Biochem.162:156-159(1987). RNA samples were analyzed by electrophoreticseparation in formaldehyde-agarose gels followed by transfer to nylonfilters. Blots were hybridized with random-primed probes generated usingeither cyr61 or GAPDH as a template. Adams et al., Nature 355:632-634(1992). The results indicated that human cyr61 mRNA is not detectablypresent in quiescent human skin fibroblasts, is abundant inlogarithmically growing and serum stimulated HSFs, and is superinducedby cycloheximide.

The analysis of RNA encoding CTGF also involved techniques that arestandard in the art. In particular, investigation of RNA encoding CTGFinvolved the isolation of total cellular RNA and Northern analyses,performed as described in U.S. Pat. No. 5,408,040, column 11, line 59,to column 12, line 14, and column 13, lines 10-29, incorporated hereinby reference. A 2.4 kb RNA was identified. The expression of CTGF washigh in the placenta, lung, heart, kidney, skeletal muscle and pancreas.However, CTGF expression was low in the liver and brain.

EXAMPLE 4 Transgenic Animals

The construction of transgenic mice bearing integrated copies ofrecombinant cyr61 sequences was accomplished using linear DNA fragmentscontaining a fusion gene. The cyr61 coding sequence was independentlyfused to the β-actin, K14, and pgk promoters, described above.Expression of cyr61 was driven by these promoters in the transgenicanimals. The fusion gene was produced by appropriate restrictionendonuclease digestions, using standard techniques. The fusion genefragments were injected into single-cell zygotes of Swiss Webster mice.The injected zygotes were then implanted into pseudopregnant females.Several litters of mice were produced in this manner. Newboms exhibitingunusual phenotypes were subjected to additional analyses. For example,neonatal transgenic mice expressing cyr61 under the pgk promoterexhibited skeletal deformities, including curly tails, immobile joints,and twisted limbs, resulting in locomotive difficulties. These micetypically were runted and died within seven days of birth. Transgenicmice expressing cyr61 under the β-actin promoter showed no obviousphenotype except that the mice were smaller. When mice bearing thetransgene were back-crossed to the in-bred strain C57BL/6, the progenymice became progressively more runted with continued back-crossing.After three to four such back-crosses, essentially no progeny survive toreproduce. Transgenic mice expressing cyr61 under the K14 promoterexhibited a form of fibrotic dermatitis. The pathology involvedexcessive surface scratching, sometimes resulting in bleeding.Transgenic organisms having knockout mutations of cyr61 can also becreated using these standard techniques, Hogan et al., Manipulating theMouse Embryo: A Laboratory Manual (Cold Spring Harbor Laboratory Press1994), and are useful as models of disease states.

EXAMPLE 5 Cyr61 Expression

Native Cyr61 is expressed in embryonic tissues and is induced in avariety of wounded tissues. See below; see also, O'Brien et al. (1992).The tissue distribution of Cyr61 was examined with rabbit anti-Cyr61polyclonal antibodies elicited using a conventional immunologicaltechnique (Harlow et al., 1987) and affinity-purified. Usingaffinity-purified anti-Cyr61 polyclonal antibodies according to theinvention, cyr61 expression was found in a variety of tissues, includingsmooth muscle, cardiomyocytes, and endothelia of the cardiovascularsystem; brain, spinal cord, ganglia and neurons, and retina of thenervous system; cartilage and bone of the skeletal system; epidermis,hair, oral epithelia, and cornea of the skin; bronchioles and bloodvessels of the lung; and placental tissues. In addition to expressionstudies directed towards native cyr61 (mRNA and protein), studies usingcyr61 transgenes, as described above, have contributed to ourunderstanding of Cyr61 expression. The use of transgene fusionscomprising the expression control sequences of cyr61 and the codingsequence of lacZ (encoding β-galactosidase) has provided a convenientcolorimetric assay for protein expression.

The colorimetric assay involves the use of5-Bromo-4-Chloro-3-Indolyl-β-D-Galactopyranoside (i.e., X-Gal) as asubstrate for β-galactosidase, the gene product of lacZ. Enzymaticcleavage of X-Gal by β-galactosidase produces an intensely coloredindigo dye useful in histochemical staining. In practice, embryonic andadult tissues subjected to analysis were dissected and fixed in 2%formaldehyde, 0.2% glutaraldehyde, 0.02% Nonidet P-40, and 0.01 sodiumdeoxycholate, in standard phosphate-buffered saline (PBS). Fixationtimes varied from 15-120 minutes, depending on the size and density oforgan or embryo samples being subjected to analysis. Subsequently,samples were rinsed in PBS and stained overnight at 37° C. in a PBSsolution containing 5 mM potassium ferrocyanide, 5 mM potassiumferricyanide, 2 mM MgCl₂, 0.02% Nonidet P-40, 0.01% sodium deoxycholateand 1 mg/ml of X-Gal (40 mg/ml in dimethylsulfoxide [DMSO]). Sampleswere then rinsed in PBS, post-fixed in 4% paraformaldehyde for 1-2hours, and stored in 70% ethanol at 4° C. until subjected to microscopicexamination. Mice containing the cyr61-lacZ transgene were used to mapthe expression profile of cyr61. The results are presented in Table Ifor embryonic tissues at day 12.5.

TABLE I Transgenic Blood Nervous Mouse Line Vessels Skeleton SystemEpidermis  1.S¹  +² − + + 2.S + + + + 3.S + +/− + + 4.T + − − NA 5.T + −− NA 6.T + +/− − NA 7.T + +/− − NA 8.T + +/− + NA ¹Transgenic lines, S-stable (established) transgenic lines; T- transient lines ²+/−Expression pattern only partially reproduced.

The results indicate that Cyr61 is expressed in a variety of embryoniccell types. Additional information has been gleaned from the ectopicexpression of Cyr61 resulting from another type of transgene fusioncomprising a heterologous expression control sequence coupled to thecoding sequence of cyr61. The control sequences, the K14 keratinpromoter, the β-actin promoter, and the phosphoglycerokinase promoter,directed the expression of Cyr61 in a pattern that differed from itsnative expression.

Transgenic mice ectopically expressing Cyr61 were routinely smaller thanwild type mice and exhibited a reduction in average life span. Moreover,these transgenic mice had abnormal hearts (i.e., thickened chamber wallswith a corresponding reduction in internal capacity) and abnormalskeletons characterized by curved spines, joints swollen to the point ofimmobility, and curly tails. Therefore, ectopic expression of Cyr61interferes with angiogenesis (blood vessel development and heartdevelopment) and chondrogenesis (skeletal development). In addition,transgenic mice carrying knockout mutations of cyr61 may be developedand tested as models of disease states associated with a lack of Cyr61activity.

A strategy for the expression of recombinant cyr61 was designed using aBaculovirus expression vector in Sf9 cells. Expression systems involvingBaculovirus expression vectors and Sf9 cells are described in CurrentProtocols in Molecular Biology §§ 16.9.1-16.12.6 (Ausubel et al., eds.,1987). One embodiment of the present invention implemented theexpression strategy by cloning the murine cyr61 cDNA into pBlueBac2, atransfer vector. The recombinant clone, along with target AcMNPV (i.e.,Autographa californica nuclear polyhedrosis virus, or Baculovirus) DNA,were delivered into Sf9 cells by liposome-mediated transfection, usingthe MaxBac Kit (Invitrogen, Inc., San Diego, Calif.) according to themanufacturer's instructions. Recombinant virus was plaque-purified andamplified by 3 passages through Sf9 cells via infection.

Conditioned medium of Sf9 insect cells infected with a baculovirusconstruct driving the synthesis of murine Cyr61 was used as a source forpurification of Cyr61 (see below). The purified recombinant Cyr61retains certain characteristics of the endogenous protein, e.g. theheparin-binding activity of Cyr61 (described below) from 3T3 fibroblastcells and had a structure similar to the endogenous protein as revealedby independent peptide profiles produced by partial proteolysis usingeither chymotrypsin or trypsin (sequencing grade; Boehringer-Mannheim,Inc., Indianapolis, Ind.).

Human cyr61 was also expressed using the baculovirus system. ASmaI-HindIlI fragment (corresponding to nucleotides 100-1649 of SEQ IDNO:3) of cyr61 cDNA spanning the entire human cyr61 open reading framewas subcloned into a pBlueBac3 baculovirus expression vector(Invitrogen). Recombinant baculovirtis clones were obtained, plaquepurified and amplified through three passages of Sf9 infection, usingconventional techniques. Infection of Sf9 cells and human Cyr61 (hCyr61)purification was performed using standard techniques, with somemodifications. Sf9 cells were maintained in serum-free Sf900-II medium(Sigma). Sf9 cells were seeded, at 2-3×10⁶ cells per 150 mm dish, inmonolayer cultures and were infected with 5 plaque forming units (PFU)of recombinant virus per cell. The conditioned medium was collected at 8and 96 hours post-infection, cleared by centrifugation (5000× g, 5minutes) and adjusted to 50 mM MES [2-(N-Morpholino)ethanesulfonicacid], pH 6.0, 1 mM PMSF (phenylmethylsulfonyl fluoride), and 1 mM EDTA.The medium was mixed with Sepharose S beads equilibrated with loadingbuffer (50 mM MES, pH 6.0, 1 mM PMSF, 1 mM EDTA, 150 mM NaCl) at a ratioof 5 ml Sepharose S beads per 500 ml of conditioned medium and theproteins were allowed to bind to the Sepharose S at 4° C. (o/n) withgentle stirring. Sepharose S beads were collected by sedimentationwithout stirring for 20 minutes and applied to the column. The columnwas washed with 6 volumes of 0.3 M NaCl in loading buffer andrecombinant human Cyr61 was eluted from the column with a step gradientof NaCl (0.4-0.8 M) in loading buffer. This procedure resulted in 3-4milligrams of purified Cyr61 protein from 500 ml of conditioned medium,and the purified Cyr61 was over 90% pure as judged by Coomassie Bluestaining of SDS-gels.

In another embodiment, the complete human cyr61 cDNA is cloned into acytomegalovirus vector such as pBK-CMV (Stratagene, LaJolla, Calif.)using the Polymerase Chain Reaction (Hayashi, in PCR: The PolymeraseChain Reaction 3-13 [Mullis et al. eds., Birkhauser 1994]) and TaqPolymerase with editing function, followed by conventional cloningtechniques to insert the PCR fragment into a vector. The expressionvector is then introduced into HUVE cells by liposome-mediatedtransfection. Recipient clones containing the vector-borne neo gene areselected using G418. Selected clones are expanded and Cyr61 expressionis identified by Reverse Transcription-Polymerase Chain Reaction (i.e.,RT-PCR; Chelly et al., in PCR: The Polymerase Chain Reaction 97-109[Mullis et al. eds., Birkhauser 1994]) or Enzyme-Linked ImmunosorbentAssays (i.e., ELISA; Stites et al., in Basic and Clinical Immunology 243[Stites et al. eds., Appleton & Lange 1991]) assays.

In other embodiments of the invention, Cyr61 protein is expressed inbacterial cells or other expression systems (e.g., yeast) using thecyr61 cDNA coding region linked to promoters that are operative in thecell type being used. Using one of these approaches, Cyr61 protein maybe obtained in a form that can be administered directly to patients,e.g., by intravenous routes, to treat angiogenic, chondrogenic, oroncogenic disorders. One of skill in the art would recognize that otheradministration routes are also available, e.g., topical or localapplication, liposome-mediated delivery techniques, or subcutaneous,intradermal, intraperitoneal, or intramuscular injection.

EXAMPLE 6 Fisp12 Expression

The expression of Fisp12, and a comparison of the expressioncharacteristics of Cyr61 and Fisp12, were investigated usingimmunohistochemical techniques. For these immunohistochemical analyses,tissue samples (see below) were initially subjected to methyl-Carnoy'sfixative (60% methanol, 30% chloroform and 10% glacial acetic acid) for2-4 hours. They were then dehydrated, cleared and infiltrated inParaplast X-tra wax at 55-56° C. for minimal duration. 7 μm thicksections were collected on poly-L-lysine-coated slides (Sigma), mountedand dewaxed. They were then treated with 0.03% solution of H₂O inmethanol for 30 minutes to inactivate endogenous peroxidase activity.After rehydration, sections were put in Tris-buffered saline (TBS: 10 mMTris, pH 7.6 and 140 mM NaCl) for 15 minutes. At that point, sectionswere blotted to remove excess TBS with paper towels and blocked with 3%normal goat serum in TBS for 10 minutes in a humid chamber. Excessbuffer was then drained and primary antibodies applied. Affinitypurified anti-Cyr61 antibodies were diluted 1:50 in 3% normal goatserum-TBS solution. Dilution for affinity-purified anti-Fisp12 antibodywas 1:25. Routine control was 3% normal goat serum-TBS, or irrelevantantibody (for example, monoclonal anti-smooth muscle cell α-actin).Specificity of staining was confirmed by incubation of anti-Cyr61 oranti-Fisp12 antibodies with an excess of the corresponding antigen onice for at least two hours prior to applying to sections. Completecompetition was observed. By contrast, cross-competition (incubation ofanti-Cyr61 antibodies with Fisp12 antigen and vice versa) did not occur.

Primary antibodies were left on sections overnight at 4° C. They werethen washed with TBS twice, and subjected to 30 minutes incubation withsecondary antibodies at room temperature. Secondary antibodies used weregoat anti-rabbit horseradish peroxidase conjugates fromBoehringer-Mannheim, Inc., Indianapolis, Ind. (used at 1:400 dilution).Sections were washed twice in TBS and chromogenic horseradish peroxidasesubstrate was applied for 5 minutes (1 mg/ml of diaminobenzidine in 50mM Tris-HCl, pH 7.2 and 0.03% H₂O₂). Sections were then counterstainedin Ehrlich's haematoxylin or in Alcian blue, dehydrated and mounted inPermount.

Mouse embryos between the neural fold (E8.5, embryo day 8.5) and lateorganogenesis (E18.5) stages of development were sectioned and subjectedto immunostaining with antigen-affinity-purified rabbit anti-Cyr61 andanti-Fisp12 antibodies. As various organs developed duringembryogenesis, the presence of Cyr61 and Fisp12 was determined. Cyr61and Fisp12 were co-localized in a number of tissues and organs. Anotable example is the placenta, where both proteins were readilydetectable. In particular, both Cyr61 and Fisp12 were found in andaround the trophoblastic giant cells, corroborating the previousdetection of cyr61 mRNA in these cells by in situ hybridization (O'Brienand Lau, 1992). Both Cyr61 and Fisp12 signals in immunohistochemicalstaining were blocked by either the corresponding Cyr61 or Fisp12antigen but not by each other, nor by irrelevant proteins, demonstratingspecificity. In general, Cyr61 and Fisp12 proteins could be detectedboth intracellularly and extracellularly.

In addition to the placenta, both Cyr61 and Fisp12 were detected in thecardiovascular system, including the smooth muscle, the cardiomyocytes,and the endothelia. Both proteins were also found in the bronchioles andthe blood vessels in the lung. Low levels of anti-Cyr61 and anti-Fisp12staining could be detected transiently in the skeletal muscle. Thisstaining is associated with connective tissue sheets, rather thanmyocytes; in this instance the staining pattern was clearlyextracellular.

A more complex pattern of distribution was found in the epidermis andthe epithelia. Both Cyr61 and Fisp12 staining could be detected in theearly, single-cell layer of embryonic epidermis, as well as in later,multilayered differentiating epidermis. Fisp12 in epidermis declined toan undetectable level by the end of gestation and remained as suchthrough adulthood, whereas Cyr61 was readily detectable in theepidermis. In the neonate, a strong staining for Fisp12 was seen in theoral epithelia where Cyr61 staining was much weaker, while Cyr61 wasfound in the upper jawbone where Fisp12 was not observed. Theanti-Fisp12 signal in the oral epithelia gradually increased andremained intense into adulthood. In the tongue, both Cyr61 and Fisp12were seen in the keratinized epithelia, although the Fisp12 stainingpattern, but not that of Cyr61, excludes the filiform papillae.

Aside from the aforementioned sites of localization, Cyr61 and Fisp12were also uniquely localized in several organ systems. For example,Cyr61, but not Fisp12, was present in skeletal and nervous systems. Asexpected from in situ hybridization results (O'Brien and Lau, 1992),Cyr61 protein was readily detected in the sclerotomal masses of thesomites, and in cartilage and bone at later stages of development. Incontrast, Fisp12 was not detectable in the skeletal system. Sincecorrelation with chondrocytic differentiation is one of the moststriking features of cyr61 expression (O'Brien and Lau, 1992), theabsence of Fisp12 in the skeletal system may underscore an importantdifference in the biological roles of Cyr61 and Fisp12. In the E14.5embryo, Cyr61 could be detected in the ventral spinal cord, dorsalganglia, axial muscle and sclerotome-derived cartilaginous vertebrae.Fisp12, however, was not detected in these tissues.

By contrast, Fisp12 was uniquely present in various secretory tissues.Beginning at E16.5, Fisp12 could be detected in the pancreas, kidneys,and salivary glands. In the pancreas, Fisp12 was strictly localized tothe periphery of the islets of Langerhans. In the kidney, strong Fisp12staining was seen in the collecting tubules and Henle's loops, regionswhere Cyr61 was not found. In the mucous-type submandibular salivarygland only collecting ducts stained for Fisp12, whereas in the mixedmucous-serous submandibular gland, both serous acini and collectingducts stained. The signal in acini was peripheral, raising thepossibility that Fisp12 is capsule-associated. In simple holocrinesebaceous glands a strong acellular Fisp12 signal was detected.

In summary, Cyr61 and Fisp12 have been co-localized in the placenta, thecardiovascular system, the lung and the skin. Neither protein wasdetected in the digestive system or the endocrine glands. Uniquelocalization of Cyr61 can be detected in the skeletal and centralnervous system, and Fisp12 is found in secretory tissues where Cyr61 isnot.

An issue closely related to protein expression concerns the metabolicfate of the expressed proteins. Members of the cysteine-rich proteinfamily have been localized. As discussed above, secreted Cyr61 is foundin the ECM and on the cell surface but not in the culture medium (Yanget al., 1991), yet secreted Fisp12 was readily detected in the culturemedium (Ryseck et al., 1991). To address the question of whether Fisp12is also ECM-associated, the fate of both Cyr61 and Fisp12 was followedusing pulse-chase experiments. Seruim-stimulated, sub-confluent NIH 3T3fibroblasts were metabolically pulse-labeled for 1 hour and chased incold medium for various times. Samples were fractionated into cellular,ECM, and medium fractions followed by immunoprecipitation to detectCyr61 and Fisp12. Both proteins have a similar short half-life ofapproximately 30 minutes in the cellular fraction, which incluides bothnewly synthesized intracellular proteins as well as secreted proteinsassociated with the cell surface (Yang and Lau, 1991). It should benoted that since Cyr61 is quantitatively secreted after synthesis andonly a minor fraction is stably associated with the ECM, the bulk ofsecreted Cyr61 is cell-surface associated (Yang and Lau, 1991).

A fraction of Cyr61 was chased into the ECM where it remained stable forseveral hours. Newly synthesized Fisp12 was also chased into the ECM,where its half-life was only about 1 hour. A larger fraction of Fisp12was chased to the conditioned medium, where no Cyr61 was detectable.Fisp12 in the conditioned medium also had a short half-life of about 2hours. Thus, whereas Cyr61 is strongly associated with the ECM, Fisp12is associated with the ECM more transiently. This result suggests thatFisp12 might be able to act at a site distant from its site of synthesisand secretion, whereas Cyr61 may act more locally.

Since many ECM proteins associate with the matrix via interaction withheparan sulfate proteoglycans, the affinity with which a protein bindsheparin might be a factor in its interaction with the ECM. The resultsof heparin binding assays, described below, are consistent with thishypothesis.

EXAMPLE 7 Protein Purification

Serum-stimulated NIH 3T3 fibroblast cells were lysed to provide a sourceof native murine Cyr61. Yang et al. Similarly, human fibroblasts are asource of native human Cyr61.

Recombinant murine Cyr61 was purified from Sf9 cells harboring therecombinant Baculovirus vector, described above, containing the completecyr61 coding sequence. Although murine Cyr61 in Sf9 cell lysates formedinsoluble aggregates as was the case with bacterial cell extracts,approximately 10% of the Cyr61 synthesized was secreted into the mediumin a soluble form. The soluble, secreted form of Cyr61 was thereforesubjected to purification.

Initially, subconfluent Sf9 cells in monolayer cultures were generatedin supplemented Grace's medium (GIBCO-BRL, Inc., Grand Island, N.Y.).Grace, Nature 195:788 (1962). The Sf9 cells were then infected with 10plaque-forming-units/cell of the recombinant Baculovirus vector,incubated for 16 hours, and fed with serum-free Grace's medium. Thesecells were expanded in serum-free Grace's Medium. The conditioned mediumwas collected 48 hours post-infection, although Cyr61 expression couldbe detected in the medium 24 hours after infection. Subsequently, theconditioned medium was cleared by centrifugation at 5000×g for 5minutes, chilled to 4° C., adjusted to 50 mM MES, pH 6.0, 2 mM EDTA(Ethylenediamine tetraacetic acid), 1 mM PMSF (Phenylmethylsulfonylfluoride) and applied to a Sepharose S column (Sigma Chemical Co., St.Louis, Mo.) at 4° C. (5 ml void volume per 500 ml medium).

The column was washed with a buffer (50 mM MES, pH 6.0. 2 mM EDTA, 0.5mM PMSF) containing 150 NaCl, and bound proteins were eluted with alinear gradient of NaCl (0.2-1.0 M) in the same buffer. The pooledfractions of Cyr61 eluted at 0.6-0.7 M NaCl as a distinct broad peak.The column fractions were 90% pure, as determined by 10% SDS-PAGEfollowed by Coomassie Blue staining or Western analysis, usingtechniques that are standard in the art. Yang et al.; see also, Sambrooket. al., supra. For Western analysis, blots were probed withaffinity-purified anti-Cyr61 antibodies as described in Yang et al.,supra. After antibody probing, Western blots were stained with ECL™(i.e., Enhanced ChemiLuminescence) detection reagents (Amersham Corp.,Arlington Heights, Ill.). Fractions containing Cyr61 were pooled,adjusted to pH 7.5 with Tris-HCl, pH 7.5, and glycerol was added to 10%prior to storage of the aliquots at −70° C. Protein concentration wasdetermined by the modified Lowry method using the BioRad protein assaykit (BioRad Laboratories, Inc., Hercules, Calif.). This purificationprocedure was repeated at least five times with similar results. Thetypical yield was 3-4 mg of 90% pure Cyr61 protein from 500 ml ofconditioned medium.

Fisp12 was purified using a modification of the Cyr61 purificationscheme (Kireeva et al., Exp. Cell Res. 233:63-77 [1997]). Serum-freeconditioned media (500 ml) of Sf9 cells infected at 10 pfu per cell werecollected 48 hours post-infection and loaded onto a 5-ml Sepharose S(Sigma Chemical Co., St. Louis, Mo.) column. After extensive washing at0.2 M and 0.4 M NaCl, bound proteins were recovered by step elution with50 mM MES (pH 6.0) containing 0.5 M NaCl. Fractions containing Fisp12 ofgreater than 80% purity were pooled, NaCl adjusted to 0.15 M and theprotein was concentrated 3-5 fold on a 0.5 ml Sepharose S column withelution of the protein at 0.6 M NaCl.

This purification scheme allowed the isolation of 1.5 mg of recombinantFisp12 protein of at least 80% purity from 500 ml of serum-freeconditioned media.

CTGF was purified by affinity chromatography using anti-PDGFcross-reactivity between CTGF and PDGF, as described in U.S. Pat. No.5,408,040, column 7, line 15, to column 9, line 63, incorporated hereinby reference.

EXAMPLE 8 Polypeptide Characterization

The murine Cyr61 protein has a M, of 41,000 and is 379 amino acids longincluding the N-terminal secretory signal. There is 91% amino acidsequence identity with the 381 amino acid sequence of the human protein.Those regions of the mouse and human proteins contributing to thesimilarity of the two proteins would be expected to participate in thebiological activities shared by the two polypeptides and disclosedherein. However, the mouse and human proteins do diverge significantlyin the central portion of the proteins, where each protein is devoid ofcysteines. See, O'Brien et al., Cell Growth & Diff. 3:645-654 (1992). Acysteine-free region in the murine Cyr61 amino acid sequence is foundbetween amino acid residues 164 to 226 (SEQ ID NO:2). A correspondingcysteine-free region is found in the human Cyr61 amino acid sequencebetween amino acid residues 163 to 229 (SEQ ID NO:4). More particularly,the mouse and human Cyr61 proteins are most divergent between Cyr61amino acids 170-185 and 210-225. Other members of the ECM signalingmolecule family of cysteine-rich proteins, e.g., Fisp12 (SEQ ID NO:6)and CTGF (SEQ ID NO:8), exhibit similar structures suggestive ofsecreted proteins having sequences dominated by cysteine residues.

Because murine Cyr61 contains 38 cysteines in the 355 amino acidsecreted portion, the contribution of disulfide bond formation to Cyr61tertiary structure was investigated. Exposure of Cyr61 to 10 mMdithiothreitol (DTT) for 16 hours did not affect the ability of Cyr61 tomediate cell attachment (see below). However, Cyr61 was inactivated byheating at 75° C. for 5 minutes, by incubation in 100 mM HCl, or uponextensive digestion with chymotrypsin. These results indicate thatmurine Cyr61 is a heat- and acid-labile protein whose activeconformation is not sensitive to reducing agents. The aforementionedstructural similarities of murine and human Cyr61 polypeptides suggeststhat human Cyr61 may also be sensitive to heat or acid, but insensitiveto reducing agents. In addition, Cyr61 is neither phosphorylated norglycosylated.

To determine if the purified recombinant murine Cyr61 described abovewas the same as native murine Cyr61, two additional characteristics ofmouse Cyr61 were determined. First, two independent protein fingerprintsof recombinant and native murine Cyr61 were obtained. Purifiedrecombinant murine Cyr61 and a lysate of serum-stimulated 3T3 cells,known to contain native murine Cyr61, were subjected to limitedproteolysis with either trypsin or chymotrypsin, and their digestionproducts were compared. Partial tryptic digests of both the recombinantprotein and cell lysate resulted in two Cyr61 fragments of approximately21 and 19 kDa. Similarly, fingerprinting of both preparations by partialchymotrypsin digestion produced stable 23 kDa fragments from recombinantmurine Cyr61 and native murine Cyr61.

Another criterion used to assess the properties of recombinant Cyr61 wasits ability to bind heparin, described below. Purified recombinantmurine Cyr61 bound quantitatively to heparin-sepharose at 0.15 M NaCland was eluted at 0.8-1.0 M NaCl. This heparin binding capacity issimilar to native murine Cyr61 obtained from serum-stimulated mousefibroblasts. Because of the similarities of the murine and human Cyr61proteins, recombinant human Cyr61 should exhibit properties similar tothe native human Cyr61, as was the case for the murine polypeptides.

The polypeptides of the invention also extend to fragments, analogs, andderivatives of the aforementioned full-length ECM signaling moleculessuch as human and mouse Cyr61. The invention contemplates peptidefragments of ECM signaling molecules that retain at least one biologicalactivity of an ECM signaling molecule, as described above. Candidatefragments for retaining at least one biological activity of an ECMsignaling molecule include fragments that have an amino acid sequencecorresponding to a conserved region of the known ECM signalingmolecules. For example, fragments retaining one or more of the conservedcysteine residues of ECM signaling molecules would be likely candidatesfor ECM signaling molecule fragments that retain at least one biologicalactivity. Beyond the naturally occurring amino acid sequences of ECMsignaling molecule fragments, the polypeptides of the invention includeanalogs of the amino acid sequences or subsequences of native ECMsignaling molecules.

ECM signaling molecule analogs are polypeptides that differ in aminoacid sequence from native ECM signaling molecules but retain at leastone biological activity of a native ECM signaling molecule, as describedabove. These analogs may differ in amino acid sequence from native ECMsignaling molecules, e.g., by the insertion, deletion, or conservativesubstitution of amino acids. A conservative substitution of an aminoacid, i.e., replacing an amino acid with a different amino acid ofsimilar properties (e.g. hydrophilicity, degree and distribution ofcharged regions) is recognized in the art as typically involving a minorchange. These minor changes can be identified, in part, by consideringthe hydropathic index of amino acids, as understood in the art. Kyle etal., J. Mol. Biol. 157:105-132 (1982). The hydropathic index of an aminoacid is based on a consideration of its hydrophobicity and charge, andinclude the following values: alanine (+1.8), arginine (−4.5),asparagine (−3.5), aspartate (−3.5), cysteine/cystine (+2.5), glycine(−0.4), glutamate (−3.5), glutamine (−3.5), histidine (−3.2), isoleucine(+4.5), leucine (+3.8), lysine (−3.9), metbionine (+1.9), phenylalanine(+2.8), proline (−1.6), serine (−0.8), threonine (−0.7), tryptophan(−0.9), tyrosine (−1.3), and valine (+4.2). It is known in the art thatamino acids of similar hydropathic indexes can be substituted and stillretain protein function. Preferably, amino acids having hydropathicindexes of ±2 are substituted.

The hydrophilicity of amino acids can also be used to revealsubstitutions that would result in proteins retaining biologicalfunction. A consideration of the hydrophilicity of amino acids in thecontext of a polypeptide permits calculation of the greatest localaverage hydrophilicity of that polypeptide, a useful measure that hasbeen reported to correlate well with antigenicity and immunogenicity.U.S. Pat. No. 4,554,101, incorporated herein by reference.Hydrophilicity values for each of the common amino acids, as reported inU.S. Pat. No. 4,554,101, are: alanine (−0.5), arginine (+3.0),asparagine (+0.2), aspartate (+3.0±1), cysteine (−1.0), glycine (0),glutamate (+3.0±1), glutamine (+0.2), histidine (−0.5), isoleucine(−1.8), leucine (−1.8), lysine (+3.0), methionine (−1.3), phenylalanine(−2.5), proline (−0.5±1), serine (+0.3), threonine (−0.4), tryptophan(−3.4), tyrosine (−2.3), and valine (−1.5). Substitution of amino acidshaving similar hydrophilicity values can result in proteins retainingbiological activity, for example immunogenicity, as is understood in theart. Preferably, substitutions are performed with amino acids havinghydrophilicity values within ±2 of each other. Both the hydrophobicityindex and the hydrophilicity value of amino acids are influenced by theparticular side chain of that amino acid. Consistent with thatobservation, amino acid substitutions that are compatible withbiological function are understood to depend on the relative similarityof the amino acids, and particularly the side chains of those aminoacids, as revealed by the hydrophobicity, hydrophilicity, charge, size,and other properties.

Additionally, computerized algorithms are available to assist inpredicting amino acid sequence domains likely to be accessible to anaqueous solvent. These domains are known in the art to frequently bedisposed towards the exterior of a protein, thereby potentiallycontributing to binding determinants, including antigenic determinants.Having the DNA sequence in hand, the preparation of such analogs isaccomplished by methods well known in the art (e.g., site-directed)mutagenesis and other techniques.

Derivatives of ECM signaling molecules are also contemplated by theinvention. ECM signaling molecule derivatives are proteins or peptidesthat differ from native ECM signaling molecules in ways other thanprimary structure (i.e., amino acid sequence). By way of illustration,ECM signaling molecule derivatives may differ from native ECM signalingmolecules by being glycosylated, one form of post-translationalmodification. For example, polypeptides may exhibit glycosylationpatterns due to expression in heterologous systems. If thesepolypeptides retain at least one biological activity of a native ECMsignaling molecule, then these polypeptides are ECM signaling moleculederivatives according to the invention. Other ECM signaling moleculederivatives include, but are not limited to, fusion proteins having acovalently modified- or C-terminus, PEGylated polypeptides, polypeptidesassociated with lipid moieties, alkylated polypeptides, polypeptideslinked via an amino acid side-chain functional group to otherpolypeptides or chemicals, and additional modifications as would beunderstood in the art. In addition, the invention contemplates ECMsignaling molecule-related polypeptides that bind to an ECM signalingmolecule receptor, as described below.

The various polypeptides of the present invention, as described above,may be provided as discrete polypeptides or be linked, e.g., by covalentbonds, to other compounds. For example, immunogenic carriers such asKeyhole Limpet Hemocyanin may be bound to a ECM signaling molecule ofthe invention.

EXAMPLE 9 Heparin Binding Assay

The heparin binding assay for native murine Cyr61, described in Yang etal., was modified for the purified recombinant murine protein.Initially, recombinant purified Cyr61 was suspended in RIPA(Radioimmunoprecipitation assay) buffer (150 mM NaCl, 1.0% NP-40, 0.5%deoxycholate, 0.1% SDS, 50 mM Tris-HCl, pH 8.0, 1 mMphenylmethylsulfonyl fluoride). Next, 200 μl of a 50% (v/v) slurry ofheparin-Sepharose CL 6B beads (Pharmacia-LKB Biotechnology, Inc.,Piscataway, N.J.) was added to 100 μl of the recombinant Cyr61 solutionand incubated for 1 hour. Under these conditions, human Cyr61 wasquantitatively bound to heparin-agarose. Application of a saltconcentration gradient in RIPA buffer resulted in the elution ofrecombinant murine Cyr61 at 0.8-1.0 M NaCl. The elution profile of therecombinant protein was similar to the elution profile for native murineCyr61.

One might expect that Fisp12 would bind heparin with lower affinity thanCyr61, as it does not interact with the ECM as strongly as Cyr61. Toexamine this possibility, metabolically labeled ([³⁵S]-cysteine; 100 μCiper 100 mm dish; ICN) cell lysates were incubated with heparin agarosebeads which were subsequently washed to remove unbound proteins. Boundproteins were eluted in increasing salt concentrations. Fisp12 from celllysates was retained on heparin agarose but was eluted by 0.2 to 0.6 MNaCl with peak elution at 0.4 M NaCl. This is in contrast to Cyr61,which was eluted at significantly higher concentrations of NaCl. Thisdifference in heparin binding is consistent with the differingaffinities of Cyr61 and Fisp12 for the ECM, suggesting that binding toheparan sulfate proteoglycans may be a primary mechanism by which bothproteins associate with the ECM.

EXAMPLE 10 Receptors

Human Cyr61, like murine Cyr61, was localized to the cell surface andECM. The localization of Cyr61 to the cell surface implicated a cellsurface receptor binding Cyr61. Consistent with that implication, thebiological effects of Cyr61 are mediated by the α_(V)β₃ integrin, orvitronectin receptor. The α_(V)β₃ integrin, in association with otherintegrins, forms protein clusters providing focal points forcytoskeletal attachment. Cyr61 induces the formation of proteinclusters, including the protein clusters containing the α_(V)β₃integrin. In addition, using an in vitro assay, the biological effectsof Cyr61, including Cyr61-induced cell adhesion and mitogenesis, wereabolished by the addition of either one of two monoclonal antibodies-LM609 (Cheresh. Proc. Natl. Acad. Sci. [USA] 84:6471-6475 [1987]) oranti-VnR 1 (Chen el al., Blood 86:2606-2615 [1995]) directed to theα_(V)β₃ integrin. This data led to the identification of the α_(V)β₃integrin as the Cyr61 receptor.

Cyr61 induction of HUVE cell adhesion, described in Example 13 below,led to an investigation of the divalent cation-sensitive cell surfacereceptors expressed by HUVE cells. The cell adhesion properties of Cyr61were used to identify the receptor, which is a divalent cation-sensitivecell surface receptor. The ability of Cyr61 to mediate cell adhesion,coupled with the strict requirement for divalent cations in the process,indicated that Cyr61 interacts with one of the divalent cation-dependentcell adhesion molecules from the integrin, selectin, or cadherinfamilies. Ruoslahti et al., Exp. Cell Res. 227:1-11 (1996). Usingwell-characterized approaches to receptor identification, a series ofinhibition studies were conducted. Inhibitors, or blocking agents, ofvarious degrees of specificity (EDTA, similar to the EGTA describedabove; inhibitory peptides bearing variants of the RGD (single letteramino acid code) integrin recognition motif, such as RGDS, SGDR, andRGDSPK (Ruoslahti, et al., Science 238:491-497 [1987], Ruoslahti. E.,Ann. Rev. of Cell and Dev. Biol. 12:698-715 [1996]); and known, specificanti-receptor antibodies) were used to identify a Cyr61 receptor. Thatreceptor was the α_(V)β₃ integrin, also known to function as thevitronectin receptor. Confirmation of that identification was obtainedby showing that antibody LM609, a specific anti-α_(V)β₃ integrinantibody, could block the effect of Cyr61 on cell adhesion. Integrinsform a large family of heterodimeric adhesion receptors, with a broadligand specificity range, involved in cell-cell and cell-matrixinteractions. Beyond their requirement for divalent cations and theirinvolvement in cell-matrix adhesion events [Hynes, R. O., Cell 69:11-25(1992)], integrins also are involved in cell migration [Damsky et al.,Curr. Opin. Cell Biol. 4:772-781 (1992); Doerr et al., J. Biol. Chem.271:2443-447 (1996)] and proliferation [Juliano et al., J. Cell Biol.,120:577-585 (1993); Plopper et al., Mol. Biol. Cell 6.1349-1365 (1995);and Clark et al., Science 268:233-239 (1995)], two additional processesassociated with Cyr61 activity. The α_(V)β₃ integrin was found to beessential for Cyr61-mediated cell adhesion.

Characterization of CTGF binding to cells has been reported to occurthrough a cell surface receptor that also interacts with PDGF-BB (the BBisoform of PDGF), as recited in U.S. Pat. No. 5,408,040, column 11, line10, to column 12, line 14, incorporated herein by reference. Theidentification of the foregoing receptors permits the design andproduction of molecules and which bind to the respective receptors toinhibit the activities of ECM molecules.

EXAMPLE 11 Anti-ECM Signaling Molecule Antibodies

Antibodies, optionally attached to a label or to a toxin as describedbelow, are also contemplated by the present invention. The availabilityof the human cyr61 cDNA sequence and the Cyr61 deduced protein sequencefacilitate the implementation of methods designed to elicit anti-Cyr61antibodies using a number of techniques that are standard in the art.Harlow et al.

In one embodiment, polyclonal antibodies directed against Cyr61 aregenerated. The generation of anti-Cyr61 antibodies specific for humanCyr61, for example, is optimized by designing appropriate antigens. Thehuman Cyr61 protein is 381 amino acids long, including the N-terminalsecretory signal. As described above, human Cyr61 exhibits a 91% aminoacid sequence identity with the 379 amino acid sequence of the mouseprotein. However, the mouse and human proteins diverge mostsignificantly in the central portion of the proteins, where they aredevoid of cysteines (see above). These sequence differences areexploited to elicit antibodies specific to the human Cyr61 by using asan antigen a peptide having a sequence derived from one of the divergentregions in the human protein, although antibodies directed to aconserved region are also contemplated by the invention.

In another embodiment of the present invention, monoclonal antibodiesare elicited using intact recombinant human Cyr61 although a fragmentmay be used. Female BALB/c mice are inoculated intraperitoneally with amixture of 0.25 ml recombinant human Cyr61 (5-50 micrograms),bacterially produced or produced in eukaryotic cells, and 0.25 mlcomplete Freund's adjuvant. Fourteen days later the injections arerepeated with the substitution of incomplete Freund's adjuvant forcomplete Freund's adjuvant. After an additional two weeks, anotherinjection of human Cyr61 in incomplete Freund's adjuvant isadministered. About two weeks after the third injection, tail bleeds areperformed and serum samples are screened for human anti-Cyr61 antibodiesby immunoprecipitation with radiolabeled recombinant human Cyr61. Abouttwo months after the initial injection, mice whose sera yield thehighest antibody titers are given booster injections of Cyr61 (5-50micrograms in incomplete Freund's adjuvant, 0.1 ml intravenously and 0.1ml intraperitoneally). Three days after the booster injection, the miceare sacrificed. Splenocytes are then isolated from each mouse usingstandard techniques, and the cells are washed and individually fusedwith a myeloma cell line, e.g., the X63Ag8.653 cell line (Harlow etal.), using polyethylene glycol, by techniques that are known in theart. Other suitable cell lines for fusion with splenocytes are describedin Harlow et al., at page 144, Table 6.2, incorporated herein byreference. Fused cells are removed from the PEG solution, diluted into acounter-selective medium (e.g., Hypoxanthine-Aminopterin-Thymidine orHAT medium) to kill unfused myeloma cells, and inoculated intomulti-well tissue culture dishes.

About 1-2 weeks later, samples of the tissue culture supernatants areremoved from wells containing growing hybridomas, and tested for thepresence of anti-Cyr61 antibodies by binding to recombinant human Cyr61bound to nitrocellulose and screening with labeled anti-immunoglobulinantibody in a standard antibody-capture assay. Cells from positive wellsare grown and single cells are cloned on feeder layers of splenocytes.The cloned cell lines are stored frozen. Monoclonal antibodies arecollected and purified using standard techniques, e.g., hydroxylapatitechromatography. In an alternative, Cyr61 peptides used as antigens, maybe attached to immunogenic carriers such as keyhole limpet hemocyanincarrier protein, to elicit monoclonal anti-Cyr61 antibodies.

Another embodiment involves the generation of antibody products againsta fusion protein containing part, or all, of human Cyr61, includingenough of the protein sequence to exhibit a useful epitope in a fusionprotein. The fusion of the large subunit of arithranilatc synthase(i.e., TrpE) to murine Cyr61, and the fusion of glutathioneS-transferase (i.e., GST) to murine Cyr61, have been used tosuccessfully raise antibodies against murine Cyr61. Yang et al. Inaddition, a wide variety of polypeptides, well known to those of skillin the art, may be used in the formation of Cyr61 fusion polypeptidesaccording to the invention.

More particularly, Yang reported a TrpE-Cyr61 fusion polypeptide thatwas expressed from a recombinant clone constructed by cloning a fragmentof the murine cyr61 cDNA containing nucleotide 456 through nucleotide951 (encoding Cyr61 amino acids 93-379) into the SacI site of the pATH1vector. Dieckman et al., J. Biol. Chem. 260:1513-1520 (1985). Therecombinant construct was transformed into a bacterial host, e.g., E.coli K12, and expression of the fusion protein was induced by additionof 25 μg/ml indoleacrylic acid to growing cultures. Subsequently, cellswere lysed and total cell lysate was fractionated by electrophoresis ona 7.5% polyacrylamide gel. The fusion protein of predicted size was theonly band induced by indoleacrylic acid; that band was eluted from thegel and used as an antigen to immunize New Zealand White rabbits(Langshaw Farms) using techniques that are standard in the art. Harlowet al. In addition to polyclonal antibodies, the invention comprehendsmonoclonal antibodies directed to such fusion proteins.

In other embodiments of the invention, recombinant antibody products areused. For example, chimeric antibody products, “humanized” antibodyproducts, and CDR-grafted antibody products are within the scope of theinvention. Kashmiri et al., Hybridoma 14:461-473 (1995), incorporatedherein by reference. Also contemplated by the invention are antibodyfragments. The antibody products include the aforementioned types ofantibody products used as isolated antibodies or as antibodies attachedto labels. Labels can be signal-generating enzymes, antigens, otherantibodies, lectins, carbohydrates, biotin, avidin, radioisotopes,toxins, heavy metals, and other compositions known in the art;attachment techniques are also well known in the art.

Anti-Cyr61 antibodies are useful in diagnosing the risk of thrombosis,as explained more fully in Example 20 below. In addition, anti-Cyr61antibodies are used in therapies designed to prevent or relieveundesirable clotting attributable to abnormal levels of Cyr61. Further,antibodies according to the invention can be attached to toxins such asricin using techniques well known in the art. These antibody productsaccording to the invention are useful in deliveringspecifically-targeted cytotoxins to cells expressing Cyr61, e.g., cellsparticipating in the neovascularization of solid tumors. Theseantibodies are delivered by a variety of administrative routes, inpharmaceutical compositions comprising carriers or diluents, as would beunderstood by one of skill in the art.

Antibodies specifically recognizing Fisp12 have also been elicited usinga fusion protein. The antigen used to raise anti-Fisp12 antibodieslinked glutathione-S-transferase (GST) to the central portion of Fisp12(GST-Fisp12), where there is no sequence similarity to Cyr61 (O'Brienand Lau, 1992). A construct containing cDNA encoding amino acids 165 to200 of Fisp12 was fused to the glutathione-S-transferase (GST) codingsequence. This was done by using polymerase chain reaction (PCR) todirect synthesis of a fragment of DNA encompassing that fragment offisp12 flanked by a 5′ BamHI restriction site and a 3′ EcoRI restrictionsite. The 5′ primer has the sequence 5′-GGGGATCTGTGACGAGCCCAAGGAC-3′(SEQ ID NO:9) and the 3′ primer has the sequence5′-GGGAATTCGACCAGGCAGTTGGCTCG-3′ (SEQ ID NO:10). For Cyr61-specificantiserum, a construct fusing the central portion of Cyr61 (amino acids163 to 229), which contains no sequence similarity to Fisp12, to GST wasmade in the same manner using the 5′ primer5′-GGGGATCCTGTGATGAAGACAGCATT-3′ (SEQ ID NO:11) and the 3′ primer5′-GGGAATTCAACGATGCATTTCTGGCC-3′ (SEQ ID NO:12). These weredirectionally cloned into pGEX2T vector (Pharmacia-LKB, lnc.) and theclones confirmed by sequence analysis. The GST-fusion protein wasisolated on glutathione sepharose 4B (Pharmacia-LKB, Inc.) according tomanufacturer's instructions, and used to immunize New Zealand whiterabbits. For affinity purifications, antisera were first passed througha GST-protein affinity column to remove antibodies raised against GST,then through a GST-Fisp12 or GST-Cyr61 protein affinity column toisolate anti-Fisp12 or anti-Cyr61 antibodies (Harlow et al., [1988]).

These antibodies immunoprecipitated the correct size Fisp12 proteinproduct synthesized in vitro directed by fisp12 mRNA. The antibodies arespecific for the Fisp12 polypeptide and show no cross-reactivity withCyr61.

Polyclonal antibodies recognizing CTGF are also known. U.S. Pat. No.5,408,040, column 7, line 41, to column 9, line 63, incorporated byreference hereinabove, reveals an immunological cross-reactivity betweenPDGF and CTGF, as described above.

EXAMPLE 12 Inhibitory Peptides

Another embodiment of the present invention involves the use ofinhibitory peptides in therapeutic strategies designed to inhibit theactivity of the Cyr61 protein. One approach is to synthesize aninhibitory peptide based on the protein sequence of Cyr61. For example,a peptide comprising an amino acid sequence that is conserved betweenmurine Cyr61 (SEQ ID NO:2) and human Cyr61 (SEQ ID NO:4) competes withnative Cyr61 for its binding sites. This competition thereby inhibitsthe action of native Cyr61. For example, administration of an inhibitorypeptide by well-known routes inhibits the capacity of Cyr61 to influencethe cascade of events resulting in blood clots, the vascularization oftumors, or the abnormal vascularization of the eye (e.g., eye disorderscharacterized by vascularization of the retina or the vitreous humor),etc. In particular, an inhibitory peptide prevents Cyr61 from inhibitingthe action of Tissue Factor Pathway Inhibitor, or TFPI, as describedbelow.

In an embodiment of the invention, inhibitory peptides were designed tocompete with Cyr61. These inhibitory peptides, like the antibodies ofthe preceding Example, exemplify modulators of Cyr61 activity, asdescribed in the context of a variety of assays for Cyr61 activity thatare disclosed herein. The peptide design was guided by sequencecomparisons among murine Cyr61, Fisp12, and Nov (an avianproto-oncogene). The amino acid sequences of several members of thisfamily are compared in FIG. 1. These types of sequence comparisonsprovide a basis for a rational design for a variety of inhibitorypeptides. Some of these designed peptides, for example peptides spanningamino acids 48-68 (SEQ ID NO:13), 115-135 (SEQ ID NO:14), 227-250 (SEQID NO:15), 245-270 (SEQ ID NO:16), and 310-330 (SEQ ID NO:17) of SEQ IDNO:2, have been synthesized. A comparison of the murine Cyr61 amino acidsequence and the human Cyr61 amino acid sequence reveals that similardomains from the human protein may be used in the design of peptidesinhibiting human Cyr61. In addition, sequence comparisons may involvethe human Cyr61 amino acid sequence; comparisons may also include thehuman homolog of Fisp12, Connective Tissue Growth Factor, alsoidentified as a member of this protein family. O'Brien et al. (1992).

Inhibitory peptides may also be designed to compete with other ECMsignaling molecules, eg., Fisp12 or CTGF, for binding to theirrespective receptors. The design of inhibiting peptides is facilitatedby the similarity in amino acid sequences among the ECM signalingmolecules. In addition, inhibitory peptide design may be guided by oneor more of the methods known in the art for identifying amino acidsequences likely to comprise functional domains (e.g., hydrophilic aminoacid sequences as external/surface protein domains; sequences compatiblewith α-helical formation as membrane-spanning domains). These methodshave been implemented in the form of commercially available software,known to those of ordinary skill in the art. See e.g., theIntelligenetics Suite of Analytical Programs for Biomolecules.Intelligenetics, Inc., Mountain View, Calif. Using these approaches,inhibitory peptides interfering with the biological activity of an ECMsignaling molecule such as Cyr61, Fisp12 or CTGF, may be designed. Withthe design of the amino acid sequence of an inhibitory peptide,production of that peptide may be realized by a variety of well-knowntechniques including, but not limited to, recombinant production andchemical synthesis. Exemplary peptides that have been shown tospecifically inhibit at least one biological activity of Cyr61 includepeptides exhibiting the “RGD” motif, or motif variants such as “RGDS,”“RGDSPK,” “GDR,” or “SGDR,” (Ruoslahti, et al., Science 238:491-497[1987], Ruoslahti, E., Ann. Rev. of Cell and Dev. Biol. 12:698-715[1996]) as described in Example 10 above.

EXAMPLE 13 Cell Adhesion

Another embodiment of the invention is directed to the use of Cyr61 tomediate cellular attachment to the extracellular matrix. Induction ofcellular adhesion was investigated using murine Cyr61, fibronectin, andbovine serum albumin (BSA). Immunological 96-well plates (Falcon brand)were coated with 50 μl of 0.1% BSA in PBS at 4° C. in the presence of0-30 μg/ml concentrations of murine Cyr61 or fibronectin. After twohours exposure to the coating solution, non-diluted immune or pre-immuneantisera (30 μl/well), or affinity-purified anti-Cyr61 antibodies wereadded. For some wells, the coating mixture was adjusted to 10 mM DTT or100 mM HCl. After 16 hours incubation, the coating solution was removedand the well surface was blocked with 1% BSA in phosphate-bufferedsaline (PBS) for 1 hour at room temperature. HUVE cells were plated inHam's complete F12K medium [GIBCO-BRL, Inc.; Ham, Proc. Natl. Acacd.Sci. (USA) 53:786 (1965)] at 5×10³-10⁴ cells/well. Cycloheximide wasadded to 100 μg/ml immediately before plating and monensin was added to1 μM 14 hours before plating. After a 2-hour incubation at 37° C., thewells were washed with PBS and attached cells were fixed and stainedwith methylene blue. The attachment efficiency was determined byquantitative dye extraction and measurement of the extract absorbance at650 nm. Oliver et al., J. Cell. Sci. 92:513-518 (1989).

HUVE cells attached poorly to dishes treated with BSA alone, but adheredwell to dishes coated with fibronectin. Murine Cyr61-coated surfacesalso supported HUVE cell attachment in a dose-dependent manner, similarto fibronectin. For example, at 1 μg/ml, Cyr61 and fibronectin yieldedA₆₅₀ values of 0.1. An A₆₅₀ value of 0.5 corresponded to the attachmentof 6×10³ cells. At the other end of the tested concentration range, 30μg/ml, Cyr61 yielded an A₆₅₀ of 0.8: fibronectin yielded an A₆₅₀ of 0.9.Cyr61 also promoted the attachment of NIH 3T3 cells, though lesseffectively than fibronectin. Cyr61-mediated cell attachment can beobserved as early as 30 minutes after plating, as visualized by lightmicroscopy.

The adhesion of HUVE cells on murine Cyr61-coated surfaces wasspecifically inhibited by anti-Cyr61 antiserum and by affinity-purifiedanti-Cyr61 antibodies, but not by pre-immune serum. In contrast,attachment of cells to fibronectin-coated dishes was not affected byeither the anti-Cyr61 antiserum or affinity-purified anti-Cyr61antibodies. These results show that enhancement of cell adhesion is aspecific activity of the Cyr61 protein. Furthermore, the Cyr61-mediatedcell attachment was insensitive to cycloheximide or monensin treatment,indicating that Cyr61 does not act by inducing de novo synthesis of ECMcomponents, stimulation of fibronectin, or collagen secretion. Rather,the data support the direct action of Cyr61 on cells in effectingadhesion. The Cyr61-mediated attachment of HUVE cells was completelyabolished by the presence of EGTA; however, attachment was restored bythe addition of CaCl₂ or MgSO₄ to the medium. These results indicatethat the interaction between Cyr61 and its cell surface receptorrequires divalent cations, consistent with the observations leading tothe identification of the α_(V)β₃ integrin as the Cyr61 receptordescribed in Example 10, above.

The ability of Cyr61 to promote cell adhesion, and the ability ofmolecules such as anti-Cyr61 antibodies to inhibit that process isexploited in an assay for modulators of cell adhesion. The assayinvolves a comparison of cell adhesion to surfaces, e.g., plastic tissueculture wells, that are coated with Cyr61 and a suspected modulator ofcell adhesion. As a control, a similar surface is coated with Cyr61alone. Following contact with suitable cells, the cells adhering to thesurfaces are measured. A relative increase in cell adhesion in thepresence of the suspected modulator, relative to the level of celladherence to a Cyr61-coated surface, identifies a promoter of celladhesion. A relative decrease in cell adhesion in the presence of thesuspected modulator identifies an inhibitor of cell adhesion.

The identification of a Cyr61 receptor led to the development of a rapidand specific ligand-receptor assay (i.e., integrin binding assay) forCyr61. Monoclonal antibody LM609 (anti-α_(V)β₃) has been described.Cheresh, 1987. Monoclonal antibody JBS5 (anti-fibronectin antibody) waspurchased from Chemicon. Anti-human and anti-bovine vitronectin antiserawere from Gibco BRL. HRP-conjugated goat anti-rabbit antibody was fromKPL. RGDSPK peptide was from Gibco BRL; RGDS and SDGR peptides were fromAmerican Peptide Company. The peptides for functional assays weredissolved in PBS at 10 mg/ml and the pH was adjusted to 7.5-8.0. withNaOH. Human plasma vitronectin was from Collaborative BiomedicalProducts.

α_(V)β₃ integrin purification from HUVE cell lysates was done asdescribed in Pytela et al., Meth. Enzynmol., 144:475-489 (1987).Briefly, 10⁸ cells were lysed in 1 ml of PBS containing 1 mM CaCl₂, 1 mMMgCl₂, 0.5 mM PMSF and 100 mM octylglucoside. The lysate was passed fourtimes through a 0.5 ml column containing RGDSPK Sepharose (prepared fromthe cyanogen bromide activated Sepharose CL 4B as described in Lam, S.C-T., J. Biol. Chem., 267:5649-5655 (1992). The column was washed with10 ml of the lysis buffer and the bound protein was eluted with 2 ml ofthe same buffer containing 1 mM RGDS peptide at room temperature. Theα_(V)β₃ integrin was dialyzed against PBS containing 1 mM CaCl₂, 1 mMMgCl₂, 5 mM octylglucoside and 0.1 mM PMSF with three changes of thedialysis buffer to remove the RGDS peptide. The protein was stored inaliquots at −70° C. The purity of the integrin was determined bySDS-PAGE under non-reducing conditions, followed by silver staining.Western blotting with anti-CD47 antibody showed that this α_(V)β₃integrin preparation does not contain any integrin-associated proteins.

The integrin binding assay was developed in accordance with thedisclosures in Brooks et al., Cell 85:683-693 (1996), and Lam, S. C-T(1992). Approximately 50 ng of the integrin in a total volume of 50 μlwere added per well of 96-well immunological Pro-Bind plates (Falcon)and incubated overnight at 4° C. Non-specific sites were blocked with 20mg/ml BSA in the same buffer and washed four times in that buffer.Treated plates were incubated with 1 μg/ml Cyr61 or 0.1 μg/mlvitronectin for 3 hours at room temperature. EDTA (5 mM), RGDS peptide(0.5 mM) and blocking antibodies were either preincubated with theimmobilized integrin for 1 hour before the addition of the proteinligand or added along with the ligand. The final dilution of the LM609ascites fluid was 1:200. Bound proteins were detected by specificpolyclonal antisera (anti-Cyr61 antiserum was diluted 1:500 andanti-vitronectin antiserum was diluted 1:1000 in PBS containing 1 mMCaCl₂, 1 mM MgCl₂, and 5 mg/ml BSA) followed by a secondaryantibody-horseradish peroxidase conjugate (1:20000 in the same buffer).Plates were rinsed four times with PBS containing 1 mM CaCl₂ and 1 mMMgCl₂ after each incubation. Horseradish peroxidase (HRP) was detectedwith an HRP immunoassay kit (Bio-Rad Laboratories). The colorimetricreaction was developed for 15-30 minutes at room temperature, stopped bythe addition of H₂SO₄, and the absorbance at 450 nm was measured. Thoseof ordinary skill in the art will understand that a variety of detectiontechniques could be employed in place of the enzyme-linked immunologicalapproach exemplified. For example, other labels such as radiolabels,fluorescent compounds and the like could be bound, e.g., covalently, toan antibody or other agent recognizing the peptide of interest such asCyr61.

The results of integrin binding assays showed that vitronectin and Cyr61bound to the immobilized integrin. Further, both Cyr61 and vitronectinbinding to α_(V)β₃ were saturable. The concentration of Cyr61 at whichsaturation was reached was significantly higher than the concentrationof vitronectin required for saturation. This difference may reflect alower affinity of α_(V)β₃ for Cyr61 compared to vitronectin, which is inagreement with the results of cell adhesion assays, which show that HUVEcells adhere to vitronectin and, more weakly, to Cyr61, in aconcentration-dependent manner (see below). The specificity of theinteraction was addressed by blocking the ligand binding site of theintegrin using any one of several techniques, including divalent cationdeprivation, RGDS peptide competition, and LM609 antibody inhibition.The interaction of both proteins (Cyr61 and vitronectin) with α_(V)β₃was inhibited by EDTA, the RGDS peptide, and the LM609 antibody. Theseproperties of the Cyr61 interaction with α_(V)β₃ were also in agreementwith the results of the cell adhesion assay and indicated that HUVE celladhesion to Cyr61 was mediated by the direct interaction of Cyr61 withthe α_(V)β₃ integrin.

In addition, Cyr61 induces focal adhesion, i.e., cell surface foci forcytoskeletal attachments. Focal adhesion is effected by cell surfaceprotein complexes or clusters. These protein clusters are complex,including a variety of receptors from the integrin family, and a varietyof protein kinases. The induction of focal adhesion by Cyr61 isreflected in the capacity of Cyr61 to induce particular members of thesecell surface protein clusters. For example, Cyr61 induces thephosphorylation of Focal Adhesion Kinase, a 125 kDa polypeptide, andPaxillin, another protein known to be involved in the focal adhesioncell surface protein complexes. Moreover, indirect immunofluorescencestudies have shown that Cyr61 is bound to a receptor (see above) infocal adhesive plaques. The plaques, in turn, are characteristic offocal adhesion protein complexes. Focal Adhesion Kinase, Paxillin, andα_(V)β₃ Integrin are co-localized to the focal adhesion plaques producedby focal adhesion complex formation induced by Cyr61. These focaladhesion protein complexes bind Cyr61 at the cell surface; the complexesalso attach internally to the cytoskeleton. Therefore, murine Cyr61, andhuman Cyr61 (see below), are, in part, adhesion molecules, acharacteristic distinguishing Cyr61 from conventional growth factors.Those of skill in the art will also recognize that the α_(V)β₃ integrincan be used, in conjunction with Cyr61,to screen for modulators of Cyr61binding to its receptor. In one embodiment, the integrin is immobilizedand exposed to either (a) Cyr61 and a suspected modulator of receptorbinding; or (b) Cyr61 alone. Subsequently, bound Cyr61 is detected,e.g., by anti-Cyr61 antibody that is labeled using techniques known inthe art, such as radiolabelling, fluorescent labeling, or the use ofenzymes catalyzing calorimetric reactions. A promoter of Cyr61 bindingto its receptor would increase binding of Cyr61 (and an inhibitor woulddecrease Cyr61), relative to the binding by Cyr61 alone.

In another embodiment of the invention, the effect of murine Cyr61 oncell morphogenesis was assessed by a cell spreading assay. PolystyrenePetri dishes were coated with 2 ml of a 10 μg/ml solution of Cyr61 orfibronectin in PBS with 0.1% BSA and treated as described above. A thirdplate was treated with BSA and served as a control. Each dish received7×10⁶ cells and was incubated for 2 hours. Cell spreading was analyzedby microscopy at 100-fold magnification. The results indicate thatmurine Cyr61 induces HUVE cell spreading to approximately the sameextent as fibronectin. The efficient attachment (see above) andspreading of cells on murine Cyr61-coated substrates indicated thatCyr61 may interact with a signal-transducing cell surface receptor,leading to a cascade of cytoskeletal rearrangements and possibleformation of focal contacts. Consequently, Cyr61 and Cyr61-relatedpolypeptides may prove useful in controlling cell adhesion, e.g., thecell adhesion events that accompany metastasizing cancer cells, organrepair and regeneration, or chondrocyte colonization of prostheticimplants, discussed below.

In contrast to mouse Cyr61 which mediated both HUVE cell attachment andmigration, hCyr61 was found to mediate cell adhesion but not spreadingof HUVE cells. Immunological plates (96-well ProBind assay plates,Falcon) were coated with 0.1-30 μg/ml hCyr61, fibronectin (Gibco BRL) orvitronectin (Gibco BRL) in phosphate-buffered saline (PBS) containing0.1% protease-free BSA (Sigma) for 16 hrs at 4° C. The wells wereblocked with 1% BSA in PBS for 1 hr at room temperature and washed withPBS. HUVE cells were harvested with 0.02% EDTA in PBS, washed twice withserum-free F12 medium and resuspended in serum-free F12. In someexperiments, fbs was added to 5-10%. Also, in experiments involvingvitronectin-coateci plates, endogenous vitronectin was removed from fbsby immunoaffinity chromatography using bovine polyclonalanti-vitronectin antibodies (Gibco). Norris et al., J. Cell Sci.95:255-262 (1990). Cells were plated at 10⁴ cells/well. After 2 hours,cells were fixed with 4% paraformaldehyde, stained with methylene blueand quantified as described. Oliver et al., J. Cell Sci. 92:513-518(1989).

Under serum-free conditions, hCyr61 mediated cell attachment but notspreading of HUVE cells. Attachment of HUVE cells to hCyr61-coatedplates was enhanced by inclusion of serum in the culture medium. In thepresence of serum, HUVE cells attached and spread on hCyr61 in a mannersimilar to that seen on fibronectin. Human Cyr61 supported HUVE celladhesion in a dose-dependent manner both under high-serum (10%) andlow-serum (0.5%) conditions. However, in the presence of 10% fbs, themaximal proportion of the cells attaching at a lower concentration ofhCyr61, and the proportion of the cells attached, was higher. HumanCyr61 was also found to cooperate with vitronectin in promoting HUVEcell adhesion and spreading. Two major cell-adhesive proteins found inmammalian sera are fibronectin and vitronectin, also known as “serumspreading factor.” For review, see Felding-Habermann et al., Curr. Opin.Cell Biol. 5:864-868 (1993). Cell attachment, spreading and growth ontissue-culture plastic depended upon vitronectin, rather thanfibronectin, in serum for the following reasons: (1) considerabledepletion of fibronectin in the batches of fbs due to “clotting” at 4°C.; and (2) inability of fibronectin to efficiently coat the plastic inthe presence of an excess amount of other serum proteins. In contrast,vitronectin coated the plastic surfaces efficiently under the sameconditions.

The ability of HUVE cells to adhere to hCyr61-coated plates in thepresence of mock-immunodepleted fbs and serum immunodepleted withanti-bovine vitronectin antibodies were compared. HUVE cells adhered tohCyr61-coated surfaces significantly better in the presence of solublevitronectin or mock-immunodepleted fbs than they did in the presence ofserum-free medium or medium supplemented with vitronectin-immunodepletedfbs. The addition of vitronectin (30 μg/ml) tovitronectin-immunodepleted serum restored the ability of HUVE cells toadhere and spread on hCyr61-coated plates to the same level observedwhen whole serum was used in the cell attachment assay. Furthermore,soluble vitronectin alone, at a concentration equal to its level in 10%serum (30 μg/ml), restored the level of cell adhesion and spreading tothe level found in the presence of 10% serum. Thus, vitronectin is anecessary and sufficient serum component contributing to HUVE celladhesion and spreading on hCyr61-coated plastic surfaces. Controlstudies showed that the effect of vitronectin was not due to itspreferential retention on the plastic dish surfaces in the presence ofhCYR61.

Additionally, HUVE cell attachment and spreading in the presence of anincreasing quantity of vitronectin was examined. The solutions forcoating the dishes contained increasing amounts of vitronectin (0-10μg/ml) with a fixed amount of hCyr61 (10 μg/ml). The results indicatedthat more cells adhered to plates coated with the two proteins thanwould have been expected by adding the individual adhesive capacities ofvitronectin and hCyr61. This non-additive increase of adhesion in thepresence of vitronectin and hCyr61 was not due to higher amounts ofvitronectin absorbed on the plastic. ELISA assay with anti-humanvitronectin antibodies showed that the amount of vitronectin adsorbed toplastic dishes exposed to the vitronectin/hCyr61 mixture did not exceedthat of vitronectin alone by more than 20%. This difference isinsufficient to explain the observed difference in cell adhesion (3-5fold in different experiments). In addition, a higher proportion of HUVEcells also adhered to the mixture of proteins when the coating solutioncontained diluted vitronectin (2.5 μg/ml) than were found to adhere todishes coated with higher concentrations of pure vitronectin (10 μg/ml)or pure hCyr61 (10 μg/ml). Thus, vitronectin and hCyr61 functionallycooperate and exert a synergistic effect on HUVE cell adhesion.

The capacity of Fisp12 to affect cell adhesion was also investigated.Fisp12 cell attachment assays were performed essentially as described(Oliver et al., [1989]). 96-well immunological plates were coated for 16hours at 4° C. with 20 μg/ml Cyr61, Fisp12 or fibronectim (Gibco BRL) inPBS containing 0.1 mg/ml BSA and blocked with 10 mg/ml BSA for 1 hour atroom temperature. HUVE cells were plated at 10⁴ cells/well in F12K mediawith 10% FBS (Hyclone Laboratories, Inc., Logan, Utah); NIH 3T3fibroblasts were plated at 3×10⁴ cells/well and Mv1Lu cells were platedat 5×10⁴ cells/well in minimal essential medium (MEM) with 10% FBS.After 1 hour incubation cells were fixed, stained with methylene blueand quantified as described (Oliver et al., [1989]). Cell spreading wasexamined on cells plated on 100 mm polystyrene petri dishes coated with2.5 ml of a 20 μg/ml solution of Cyr61, Fisp12 or fibronectin. 10⁷ cellswere plated on each dish and cell spreading was analyzed 90 minutesafter plating by microscopy at 100× magnification.

The results indicated that Fisp12, as well as Cyr61, when coated onplastic dishes, promoted the attachment of three different cell types:HUVE cells, NIH 3T3 fibroblasts, and mink lung epithelial (Mv1Lu) cells.These cells attached poorly to uncoated plastic dishes or plastic dishescoated with bovine serum albumin, but attached significantly better todishes coated with either fibronectin, Cyr61, or Fisp12. The ability ofeither Cyr61 or Fisp12 to mediate cell attachment is comparable to thatof fibronectin for all three cell types. While the ability of Cyr61 tomediate cell attachment was previously demonstrated for fibroblasts andendothelial cells (Kireeva et al., Mol. Cell Biol. 16:1326-1334 [1996]),these studies show cell attachment activity for both Fisp12 and Cyr61 inepithelial cells in addition to endothelial cells and fibroblasts.

Like cell attachment to fibronectin and Cyr61 (Kireeva et al., [1996]),Fisp12-mediated cell attachment was inhibited when EDTA was added to theculture medium. This inhibition was completely abolished by the additionof excess MgCl₂, indicating a requirement for divalent cations inFisp12-mediated cell attachment. In addition to cell attachment, Fisp12also promotes cell spreading. Similar cell spreading was found when NIH3T3 cells were plated on dishes coated with either fibronectin, Cyr61 orFisp12, but not BSA. Endothelial and epithelial cells also spread whenplated on fibronectin, Cyr61, or Fisp12.

EXAMPLE 14 Migration of Fibroblasts

Cyr61 also affects chondrocytes, e.g., fibroblasts involved in skeletaldevelopment. In particular, Cyr61 influences the development, andperhaps maintenance, of cartilage, in contrast to the variety ofgrowth-related proteins that exclusively influence development andmaintenance of the bony skeleton. The chemotactic response of NIH 3T3cells to murine Cyr61 was examined using a modified Boyden chamber(Neuroprobe Inc., catalog no. AP48). Grotendorst, Meth. Enzymol.147:144-152 (1987). Purified Cyr61 protein was serially diluted in DMEMcontaining bovine serum albumin (BSA; 0.2 mg/ml) and added to the lowerwell of the chamber. The lower well was then covered with acollagen-coated polycarbonate filter (8 μm pore diameter; NucleoporeCorp., Pleasanton, Calif.). Cells (6×10⁴) were then loaded into theupper well. After 5 hours incubation (10% CO₂, 37° C.), the filter wasremoved and the cells were fixed and stained using Wright-Giemsa stain(Harleco formulation; EM Diagnostic Systems, Gibbstown, N.J.). Cellsfrom the upper surface of the filter were then removed by wiping with atissue swab. The chemotactic response was determined by counting thetotal number of migrating cells detected in ten randomly selectedhigh-power microscopic fields (400-fold magnification) on the lowersurface of the filter. Duplicate trials were performed for eachexperiment and the experiment was repeated three times to ensurereproducibility of the data.

NIH 3T3 cells responded to Cyr61 as a chemotactic factor in adose-dependent manner in the Boyden chamber assay. Without Cyr61,approximately 4.8 cells had migrated per high-power field. In thepresence of 0.5 μg/ml murine Cyr61, about 5.2 cells were found in eachfield. As the concentration of Cyr61 was raised to 1, 5 and 10 μg/ml,the average number of migrating cells detected per field rose to 7.5,18.5, and 18.7. Thus, murine Cyr61 acts as a chemoattractant forfibroblasts. The optimal concentration for the chemotactic activity ofCyr61 is 1-5 μg/ml in this assay; this concentration range is consistentwith the reported ranges at which other ECM molecules provide effectivechemotactic stimulation. For example, Thrombospondin, at 5-50 μg/ml, hasa chemotactic effect on endothelial cells (Taraboletti et al., J. CellBiol. 111:765-772 (1990); fibronectin also functions as a chemotacticagent at 1-30 μg/ml (Carsons et al., Role of Fibronectin in RheumaticDiseases, in Fibronectin [Mosher, ed., Academic Press 1989]; Carsons etal., Arthritis. Rheum. 28:601-612 [1985]) as determined using similarBoyden chamber assays. The human Cyr61 polypeptide may be used tochemoattract fibroblasts in a manner analogous to murine Cyr61. Inexperiments analogous to the studies of NIH 3T3 cell migration inresponse to murine Cyr61, the migration of 1064SK human skin fibroblastsin response to wild-type human Cyr61 , and human Cyr61 NT (a Cyr61polypeptide fragment described in greater detail in Example 29), wasdetermined. Cyr61 induced migration of the human fibroblast cells atcomparable levels to the murine Cyr61 levels inducing NIH3T3 cellchemotaxis. The human Cyr61 NT induced 1064SK cell migration at levelscomparable to the effective levels of wild-type human Cyr61. Antibodystudies showed that an antibody specific for the α_(V) integrin subunitblocked fibroblast migration in the presence of Cyr61 polypeptides,while an antibody (i.e., GoH3) specific for the α₆ integrin subunit didnot block migration. Further investigation revealed that a monoclonalantibody specifically recognizing the α_(V)β₅ integrin inhibitedCyr61-induced human fibroblast migration, but a monoclonal antibodyspecifically recognizing the integrin α_(V)β₃ did not inhibit thatmigration. Thus, integrin α_(V)β₅ mediates the fibroblast migrationresponse to Cyr61 polypeptides. These results contrast with the resultsof antibody studies analyzing the chemotactic response of endothelialcells to Cyr61 polypeptides, which is dependent on integrin α_(V)β₃.Human CTGF has also been reported to induce the migration of non-humanmammalian cells such as NIH 3T3 cells (mouse fibroblasts) and BASM cells(bovine aortic smooth muscle cells), as described in U.S. Pat. No.5,408,040, column 7, line 65 to column 11, line 7, incorporated hereinby reference.

Thus, one aspect of the invention is a method of screening formodulators of cell migration, such as fibroblast cell migration. Ingeneral, the method involves exposing cells that preferably present atleast one suitable integrin receptor to a Cyr61 polypeptide in thepresence or absence of a potential, or suspected, modulator. Subsequentmeasurement of relative cell migration rates (in the presence andabsence of the potential modulator) identifies modulators ofCyr61-induced cell migration. A variety of small chemicals, bothinorganic and organic, as well as various peptides, are expected to bepotential modulators. Examples include mannose and its derivative,mannose-6-phosphate, with the latter expected to function as a modulatorof Cyr61-induced cell migration by inhibition. The potential modulatorsscreened by methods of the invention can have any of a wide variety ofstructures and may be tested individually or as part of a moresystematic approach using any of the chemical or peptide libraries knownin the art.

In an alternative embodiment, an assay for modulators of cell migration,such as the migration of chondrocytes, involves a combination of asuspected modulator of cell migration and Cyr61 being added to the lowerwell of a Boyden chamber. As a control, Cyr61 is separately added to thelower well of another Boyden chamber. Relative cell migrations are thenmeasured. An increase in cell migration in the presence of the suspectedmodulator relative to cell migration in response to Cyr61 aloneidentifies a promoter of chondrocyte cell migration, while a relativedecrease in cell migration in the presence of the suspected modulatoridentifies an inhibitor.

EXAMPLE 15 Migration of Endothelial Cells—In Vitro Assays

The end product of in vitro angiogenesis is a well-defined network ofcapillary-like tubes. When cultured on gel matrices, e.g., collagen,fibrin, or Matrigel gels, endothelial cells must first invade the matrixbefore forming mature vessels. (Matrigel is a complex mixture ofbasement membrane proteins including laminin, collagen type IV,nidogen/entactin, and heparan sulfate proteoglycan, with additionalgrowth factors. Kleinman el al., Biochem. 25:312-318 (1986). Theinvasive structures are cords which eventually anastomose to form thevessel-like structures. The angiogenic effect of human Cyr61 onconfluent monolayers of human umbilical vein endothelial cells isassessed by seeding the cells onto three-dimensional collagen or fibringels, in the presence or absence of Cyr61. HUVE cels do notspontaneously invade such gels but do so when induced by agents such astumor promoters.

Collagen gels were prepared by first solubilizing type I collagen(Collaborative Research, Inc., Bedford, Mass.) in a sterile 1:1000 (v/v)dilution of glacial acetic acid (300 ml per gram of collagen). Theresulting solution was filtered through sterile triple gauze andcentrifuged at 16,000× g for 1 hour at 4° C. The supernatant wasdialyzed against 0.1× Eagle's Minimal Essential Medium (MEM; GIBCO-BRL,Inc.) and stored at 4° C. Gels of reconstituted collagen fibers wereprepared by rapidly raising the pH and ionic strength of the collagensolution. The pH and ionic strength adjustments were accomplished byquickly mixing 7 volumes of cold collagen solution with one volume of10× MEM and 2 volumes of sodium bicarbonate (11.76 mg/ml) in a sterileflask. The solution was kept on ice to prevent immediate gelation. Thecold mixture was dispensed into 18 mm tissue culture wells and allowedto gel for 10 minutes at 37° C.

Fibrin gels were prepared by dissolving fibrinogen (Sigma Chemical Co.St. Louis, Mo.) immediately before use in calcium-free MEM to obtain afinal concentration of 2.5 mg of protein/ml. Clotting was initiated byrapidly mixing 1.35 ml of fibrinogen solution with 15 μl of 10× MEMcontaining 25 U/ml thrombin (Sigma Chemical Co.) in a plastic tube. Themixture was transferred immediately into 18 mm tissue culture wells andallowed to gel for about 2 minutes at 37° C.

In some wells, Cyr61 was mixed into the gel matrix before gelation(final concentration 10 μg/ml), while in other wells, Cyr61 was not inthe gel matrix but was added as part of the nutrient medium (similarrange of concentrations as in the matrix) after the cells reachedconfluency. HUVE cells were seeded onto the gel matrix surface at 5×10⁴cells per well in Ham's F12K medium [GIBCO-BRL, Inc.] containing 10%fetal bovine serum, 100 μg/ml heparin, and 30 μg/ml endothelial cellgrowth factor. When the cells reached confluency, the medium wasremoved, the cells were rinsed with PBS, and fresh medium withoutendothelial cell growth factor was supplied. Some cultures receivedpurified recombinant Cyr61, while others received Cyr61 and polyclonalanti-Cyr61 antibodies. Thus, the variety of cultures at confluencyincluded: a) cultures with no Cyr61; b) cultures with Cyr61 within thematrix; c) cultures with Cyr61 supplementing the medium; and d) cultureswith Cyr61 supplementing the medium along with polyclonal anti-Cyr61antibodies.

Invasion of the gel matrix was quantified about 4-7 days after treatmentof the confluent cultures. Randomly selected fields measuring 1.0 mm×1.4mm were photographed in each well under phase-contrast microscopy with aZeiss Axiovert inverted photomicroscope. Photographs were taken at asingle level beneath the surface monolayer. Invasion was quantified bymeasuring the total length of all cell cords that penetrated beneath thesurface monolayer. Results were expressed as the mean length in micronsper field for at least 3 randomly selected fields from each of at least3 separate experiments.

In order to examine the network of cords within the matrix forcapillary-like tube formation, cultures were fixed in situ overnightwith 2.5% glutaraldehyde and 1% tannic acid in 100 mM sodium cacodylatebuffer, pH 7.4. Cultures were then washed extensively in 100 mM sodiumcacodylate buffer, pH 7.4. The gels were cut into 2 mm×2 mm fragments,post-fixed in 1% osmium tetroxide in veronal acetate buffer (to minimizetissue swelling; see Hayat, in Principles and Techniques of ElectronMicroscopy,: Biological Applications 1:38 [Litton EducationalPublishing, Inc. 1970]) for 45 minutes, stained en bloc with 0.5% uranylacetate in veronal buffer for 45 minutes, dehydrated by exposure to agraded ethanol series, and embedded in Epon in flat molds. Semi-thinsections were cut perpendicular to the culture plane with anultramicrotome, stained with 1% toluidine blue, and photographed undertransmitted light using an Axiophot photomicroscope (Zeiss).

In an alternative embodiment, a suspected modulator of angiogenesis iscombined with Cyr61 and the combination is added before, or after,formation of a gel. In this embodiment, a control is established byusing Cyr61 alone. The migration of cells in response to the suspectedmodulator and Cyr61 is then compared to the migration of cells inresponse to Cyr61 alone. A promoter or positive effector will increasecell migration while an inhibitor or negative effector will decreasecell migration.

In an alternative in vitro assay for angiogenic activity, an assay forendothelial cell migration was developed. This chemotaxis assay has beenshown to detect the effects of Cyr61 concentrations on the order ofnanograms per milliliter. Primary Human Microvascular Endothelial Cells(HMVEC PO51, Clonetics, San Diego, Calif.) were maintained in DME with10% donor calf serum (Flow Laboratories, McLean, Va.) and 100 μg/mlendothelial celi mitogen (Biomedical Technologies Inc., Stoughton,Mass.). The cells were used between passages 10 and 15. To measuremigration, cells were starved for 24 hours in DME containing 0.1% BSA,harvested, resuspended in DME with 0.1% BSA, and plated at 1.75×10⁴cells/well on the lower surface of a gelatinized 0.5 μm filter(Nucleopore Corporation, Pleasanton. Calif.) in an inverted modifiedBoyden chamber. After 1-2 hours at 37° C., during which time the cellswere allowed to adhere to the filter, the chamber was reverted to itsnormal position. To the top well of separate chambers, basic FibroblastGrowth Factor (a positive control), Cyr61, or a negative controlsolution (conditioned medium known to lack chemoattractants or DME plusBSA, see below) was added at concentrations ranging from 10 ng/ml to 10μg/ml. Chambers were then incubated for 3-4 hours at 37° C. to allowmigration. Chambers were disassembled, membranes fixed and stained, andthe number of cells that had migrated to the top of the filter in 3high-powered fields was determined. Tolsma et al., J. Cell. Biol.122:497-511 (1993) (incorporated by reference), and references citedtherein. DME with 0.1% BSA was used as a negative control and eitherbFGF (10 ng/ml) or conditioned media from angiogenic hamster cell lines(20 μg/ml total protein) were used as positive controls. Rastinejad etal., Cell 56:345-355 (1989). Each sample was tested in quadruplicate(test compound such as Cyr61, positive control, conditioned medium as anegative control, and DME plus BSA as a negative control) in a singleexperiment; experiments were repeated at least twice.

To allow comparison of experiments performed on different days,migration data is reported as the percent of maximum migration towardsthe positive control, calculated after subtraction of backgroundmigration observed in the presence of DME plus BSA. Test compounds thatdepressed the random movement of endothelial cells showed a negativevalue for the percent migration. Very high concentrations ofthrombospondin (TSP) caused endothelial cells to detach from themembrane. Detachment was detected by counting cells on the lower face ofthe membrane. When cell loss exceeded 10%, the number of migrated cellswas corrected for this loss. The results indicate that 0.01-10 μg/mlbFGF induced the migration of a constant 92 cells per three high-poweredmicroscope fields Migration in the presence of Cyr61 revealed a greaterdependence on concentration. At 10 ng/ml, Cyr61 induced an average of 64cells to migrate per three high-powered fields examined. At 100 ng/mlCyr61, approximately 72 cells were found in three fields; at 1 μg/mlCyr61, a peak of 87 cells had migrated; at approximately 7 μg/ml Cyr61,about 61 cells were observed; and at 10 μg/ml Cyr61, approximately 57cells were found to have migrated. The negative control revealed aconstant basal level of endothelial cell migration of 53 cells per threehigh-powered microscope fields. In addition to these results, there is aperfect correlation of the results from this in vitro assay and theresults from the in vivo cornea assay, described below.

To monitor toxicity, endothelial cells were treated with each of thetested compounds at a range of concentrations, under conditionsidentical to those used in the migration assay. Cells were then stainedwith Trypan blue and cells excluding Trypan blue were counted. Theresults showed that cells remained viable and that the inhibition ofmigration could not be attributed to toxicity. Where relevant,endothelial cells were pretreated for 36-48 hours with peptides at 20 μMin DME with 0.1% BSA before use in the migration assays. Toxicity wasalso tested over these time frames and found to be negligible.

The ability of Cyr61 to induce matrix invasion and tube formation byHUVE cells, as well as the ability of Cyr61 to induce humanmicrovascular endothelial cells to migrate, demonstrates the angiogenicproperties of this protein. It is anticipated that other members of theECM signaling molecule family of cysteine-rich proteins, such as Fisp12and CTGF, have similar properties that may be used in methods of theinvention for screening for, and modulating, angiogenic conditions. Inparticular, one of ordinary skill in the art understands that an invitro assay for angiogenic inhibitors involves the assay describedabove, including an effective amount of Cyr61, with and without thecandidate inhibitor.

EXAMPLE 16 Migration of Endothelial Cells—An In Vitro Assay ForAngiogenesis Inhibitors

The inclusion of an effective amount of an ECM signaling molecule, suchas Cyr61, in the in vitro migration (i.e., chemotaxis) assay describedin the preceding Example, provides an assay designed to detectinhibitors of ECM signaling molecules and angiogenesis. Because of thecrucial role of neovascularization in such processes as solid tumorgrowth and metastasis, the development of assays to detect compoundsthat might antagonize these processes would be useful.

The above-described in vitro migration assay was adapted to include anECM signaling molecule, Cyr61. Cyr61 was included at 1 μg/ml, which wasfound to be the optimal dosage in titration studies. As in the precedingExample, human microvascular endothelial cells (Clonetics) were used. Inone series of assays, several carbohydrates and carbohydrate derivativeswere analyzed. These compounds included 10 mM mannose, 10 mMmannose-6-phosphate, and 10 mM galactose. Results of these assays showedthat Cyr61 plus mannose yielded approximately 73 cells per set of threehigh-powered microscope fields (see above). Cyr61 plus galactose inducedthe migration of approximately 74 cells per set of three high-poweredfields. However, Cyr61 plus mannose-6-phosphate yielded approximately 2migrating cells for each set of three high-powered fields examined.Control experiments demonstrate that the inhibition of Cyr61 activity bymannose-6-phosphate is specific.

The angiogenic activity of basic FGF (10 ng/ml) was also tested, asdescribed above, with and without mannose-6-phosphate. In the presenceof 10 mM mannose-6-phosphate, bFGF induced 51 cells per set of threehigh-powered fields to migrate; in its absence, bFGF induced themigration of approximately 52 cells. However, when either Cyr61 orInsulin Growth Factor II (IGF-II) were tested, mannose-6-phosphatereduced the number of migrating cells from approximately 48 or 47 cells,respectively, to approximately 12 or 11 cells, respectively. The effectof mannose-6-phosphate on IGF II activity was anticipated becausemannose-6-phosphate is known to compete with IGF II for their commonreceptor (the IGF II receptor). Thus, mannose-6-phosphate specificallyinhibits the chemotactic activity of Cyr61 on human endothelial cells.Moreover, because there is an essentially perfect correlation betweenthe in vitro migration assay and the in vivo angiogenesis assay,described below, mannose-6-phosphate has been identified as an inhibitorof angiogenesis based on the results of the assay disclosed herein.Accordingly, the invention contemplates a method of inhibitingangiogenesis comprising the step of administering an inhibitor theangiogenic activity of Cyr61 such as mannose-6-phosphate. Assays such asthat described above may also be used to screen for other inhibitors ofangiogenesis which may be useful in the treatment of diseases associatedwith angiogenesis such as cancer, and diseases of the eye which areaccompanied by neovascularization.

In an embodiment of the invention, a method of screening for modulatorsof angiogenesis involves a comparative assay. One set of conditionsinvolves exposure of cells to a combination of Cyr61 and a suspectedmodulator of cell migration. As a control, a parallel assay is performedthat exposes cells to Cyr61 alone. A promoter of cell migration elevatesthe rate of in vitro cell migration relative to the rate of migration inthe presence of Cyr61 alone; the converse is true for an inhibitor ofthe chemoattracting ability of Cyr61.

EXAMPLE 17 Migration of Endothelial Cells—An In Vivo Assay

An in wivo assay for endothelial cell migration has also been developed.In general, the assay protocol is consistent with the disclosure ofTolsma et al., 1993. To assess angiogenesis associated with theformation of granulation tissue (i.e., the newly formed, proliferative,fibroblastic dermal tissue around wounds during healing), spongeimplants were used as previously described (Fajardo, et al., Lab.Invest. 58:718-724 [1988]). Polyvinyl-alcohol foam discs (10-mmdiam×1-mm thick) were prepared by first removing a 2-mm diameter centralcore of sponge. PBS or an RGDS peptide (other possible test compoundsinclude fragments of Cyr61, RGDS peptide, small molecules such asmannose-6-phosphate) at 100 μM were added to the sponge core which wasthen coated with 5 μl of sterile Hydron (Interferon Sciences, NewBrunswick, N.J.). After solidifying, the coated core was returned to thecenter of the sponge which was then covered on both sides with 5 μmfilters and secured in place with glue (Millipore Corp., Bedford,Mass.). One control and one test disc were then implanted subcutaneouslyin the lower abdomen of anesthetized Balb/c female mice wheregranulation tissue could invade the free perimeter of the disc. Woundswere closed with autoclips and animals left undisturbed untilsacrificed.

Quantitative estimates of thymidine incorporation in situ intoendothelial cells in the discs were obtained as previously described(Polverini. et al., J. Immunol. 118:529-532 [1977]). Sponge implantswere evaluated at days 5, 7, 10, and 14 after implantation. Thirtyminutes before sacrifice, mice were injected with a solution containing[³H]-thymidine in saline (specific activity 6.7 Ci/mM; New EnglandNuclear/Du Pont, Wilmington, Del.) to a level of 1 μCi per gram of bodyweight. Sponges were removed and facially embedded to provide a uniformsection of the entire circumference. Tissues were fixed in 10% neutralbuffered formalin, dehydrated through a graded series of alcohols, andembedded in glycol methacrylate (Polysciences, Miles, Ill.).Autoradiograms were prepared by dipping sections mounted on acid-cleanedglass slides into NTB type 2 emulsion (Eastman Kodak). After exposurefor 4 weeks at 4° C., autoradiographs were developed in half strengthD-19 developer, fixed in Kodak Rapid Fixer, and stained with hematoxylinand eosin. Quantitation of endothelial cell labeling was performed bycounting all endothelial cells that lined capillaries and venulesextending from the periphery to the center of the sponge by rectilinearscanning under oil immersion (×1,000). A total of 500-700 endothelialcells were counted in each of two sponges containing either PBS, TSP, orpeptide fragments (i.e., thrombospondin fragments). Cells wereconsidered labeled if five or more grains were detected over thenucleus. The percentage of labeled cells was calculated and a chi-squareanalysis of data derived from control and experimental sponges wasperformed.

The results of the foregoing assay established that thrombospondinfragments could inhibit the process of angiogenesis. More generally, oneof ordinary skill in the art would appreciate that the scope of thepresent invention extends to such in vitro assays for suspectedmodulators of ECM signaling molecule activities, such as the chemotacticability of Cyr61 to induce cell migration. As vith other assays of theinvention, a comparative assay involves exposure of cells, in vivo, to asponge laden with Cyr61 in the presence or absence of a suspectedmodulator of Cyr61 activity. Following implantation, incubation, andremoval, the relative rates of cell migration are determined. A promoterof Cyr61 activity will increase the rate of cell migration relative tocell migration induced by Cyr61 alone; an inhibitor will decrease therate of cell migration relative to the level ascribable to Cyr61 alone.

EXAMPLE 18 Mitogen Potentiation

In another aspect of the invention, murine Cyr61 enhanced the mitogeniceffect of growth factors on fibroblasts and endothelial cells. When NIH3T3 fibroblasts or HUVE cells were treated with a non-saturating dose ofeither basic Fibroblast Growth Factor (bFGF) or Platelet-Derived GrowthFactor (PDGF-BB), the addition of murine Cyr61 or human Cyr61polypeptides (wild-type Cyr61 and Cyr61 NT), as well as Fisp12/CTGF,significantly increased the incorporation of radiolabeled thymidinecompared to cells treated with the growth factors alone. The thymidineincorporation assay is a standard technique for determining whethercells are actively glowing by assessing the extent to which the cellshave entered the S phase and are synthesizing DNA. The Cyr61 enhancementof bFGF- or PDGF-BB-induced thymidine incorporation was dose-dependent,requiring a minimum concentration of 0.5-1.0 μg/ml of recombinantprotein for either cell type. Cyr61 polypeptides are expected to haveanalogous enhancing or potentiating effects on the ability of othergrowth factors (e.g.,PDGF-BB) to induce the mitogenesis of endothelialcells, fibroblasts, and other mammalian cell types as measured, e.g., bythymidine incorporation (i.e., DNA synthesis). The enhancement of DNAsynthesis by Cyr61 was inhibited by the addition of specific anti-Cyr61antiserum.

More specifically, NIH 3T3 fibroblast cells were plated on 24-wellplates at 3×10⁴ cells/well and grown in DMEM with 10% fetal bovine serum(Intergen Co., Purchase, N.Y.) for 3-4 days and incubated with mediumcontaining 0.2% FBS for the following 48 hours. The following compounds,at the parenthetically noted final concentrations, were then added tothe plated cells in fresh DMEM containing 0.2% fbs and [³H]-thymidine (1μCi/ml final concentration; ICN Biomedicals, Inc., Costa Mesa, Calif.):bFGF (15 ng/ml), PDGF-BB (30 ng/ml), and murine Cyr61 (0.5-5 μg/ml).These compounds were added to individual plates according to thefollowing pattern: 1) no supplementation; 2) murine Cyr61; 3) bFGF; 4)murine Cyr61 and bFGF; 5) PDGF-BB; and 6) murine Cyr61 and PDGF. After18-20 hours of incubation, cells were washed with PBS and fixed with 10%trichloroacetic acid. DNA was dissolved in 0.1 N NaOH and thymidineincorporation was determined. The results indicated that murine Cyr61,in the absence of a growth factor, did not stimulate DNA synthesis asmeasured by tritiated thymidine incorporation. Without any supplements,3T3 cells incorporated approximately 1.8×10⁴ cpm of [³H]-thymidine, inthe presence or absence of Cyr61. Cells exposed to bFGF aloneincorporated about 1.2×10⁵ cpm; cells contacting bFGF and murine Cyr61incorporated 2×10⁵ cpm. Cells receiving PDGF-BB incorporated about1.2×10⁵ cpm; and cells exposed to PDGF-BB and murine Cyr61 incorporatedapproximately 2.4×10⁵ cpm. Therefore, murine Cyr61 did not function as amitogen itself, but did potentiate the mitogenic activity of bFGF andPDGF-BB, two known growth factors.

The ability of murine Cyr61 to potentiate the mitogenic effect ofdifferent levels of bFGF also revealed a threshold requirement for thegrowth factor. Human umbilical vein endothelial cells were platedessentially as described above for 3T3 cells and exposed to a constantamount of murine Cyr61; controls received no Cyr61. Different plateswere then exposed to different levels of bFGF, comprising a series ofbFGF concentrations ranging from 0-10 ng/ml. Following culture growth inthe presence of [³H]-thymidine for 72 hours, cells exposed to 0-0.1ng/ml of bFGF exhibited a baseline level of thymidine incorporation(approximately 4×10² cpm), in the presence or absence of Cyr61. At 1ng/ml bFGF, however, HUVE cells increased their thymidine incorporationin the presence of bFGF to 6×10² cpm; in the presence of 1 ng/ml bFGFand murine Cyr61, HUVE cells incorporated 1.3×10³ cpm. At 10 ng/ml bFGF,cells exposed to bFGF incorporated about 1.8×10³ cpm thymidine; cellsreceiving 10 ng/ml bFGF and Cyr61 incorporated approximately 6.1×10³cpm.

The capacity of murine Cyr61 to potentiate the mitogenic activity ofbFGF was verified by a thymidine incorporation assay involving HUVEcells and various combinations of bFGF, Cyr61, and anti-Cyr61antibodies. Cells were plated and grown as described above. Thefollowing combinations of supplements (final plate concentrations notedparenthetically) were then pre-incubated for 1 hour before addition toindividual plates: 1) pre-immune antiserum (3%); 2) bFGF (15 ng/ml) andpre-immune antiserum (3%); 3) pre-immune antiserum (3%) and Cyr61 (4μg/ml); 4) pre-immune antiserum (3%), Cyr61 (4 μg/ml), and bFGF (15ng/ml); 5) anti-Cyr61 antiserum (3%); 6) anti-Cyr61 antiserum and bFGF(15 ng/ml); 7) anti-Cyr61 antiserum (3%) and Cyr61 (4 μg/ml); and 8)anti-Cyr61 antiserum (3%), Cyr61 (4 μg/ml), and bFGF (15 ng/ml).

Following incubation in the presence of [³H]-thymidine as describedabove, cells exposed to pre-immune antiserum incorporated about 2×10²cpm thymidine; cells contacting pre-immune antiserum and bFGFincorporated 1.3×10³ cpm; cells receiving pre-immune antiserum and Cyr61incorporated 1×10² cpm; cells receiving pre-immune antiserum, Cyr61, andbFGF incorporated 3.6×10³ cpm; cells exposed to anti-Cyr61 antiserumincorporated 2×10² cpm; cells receiving anti-Cyr61 antiserum and bFGFincorporated about 1.3×10³ cpm; cells contacting anti-Cyr61 antiserumand Cyr61 incorporated about 1×10², and cells receiving anti-Cyr61antiserum, Cyr61, and bFGF incorporated 1×10³ cpm. These resultsindicate that pre-immune antiserum had no effect on Cyr61-inducedpotentiation of bFGF mitogenic activity. Anti-Cyr61 antiserum, however,completely abolished the potentiation of bFGF by Cyr61. Moreover, theeffect of anti-Cyr61 antiserum was specific to Cyr61-induced mitogenicpotentiation because anti-Cyr61 antiserum had no effect on the mitogenicactivity of bFGF per se. Therefore, Cyr61 can be used as a reagent toscreen for useful mitogens.

Additional antibody studies using integrin-specific monoclonalantibodies showed that from an entire panel of specific anti-integrinantibodies, only an antibody (LM609) specific for integrin α_(V)β₃inhibited the induction of DNA synthesis by Cyr61 polypeptides,including wild-type Cyr61 and Cyr61 NT. Thus, integrin α_(V)β₃ isrequired for Cyr61-induced mitogenesis, although it is not involved infibroblast adhesion (see Example 19).

DNA synthesis for HUVE cells and NIH 3T3 fibroblasts was measured bythymidine incorporation as described previously (Kireeva et al., [1996])with minor modifications. HUVE cells were grown in 24-well plates to asubconfluent state, serum-starved for 24 hours and treated with F12Kmedium containing 10% fetal bovine serum (FBS), 1 μCi/ml [³H]-thymidineand 10 ng/ml basic Fibroblast Growth Factor (bFGF) (Gibco-BRL, Inc.)with various concentrations of Cyr61 and Fisp12 as indicated. NIH 3T3fibroblasts were grown to subconlfluence, serum-starved for 48 hours,and treated with Minimal Essential Medium (MEM) containing 0.5% FBS, 1μCi/ml [³H]-thymidine, bFGF and various concentrations of Cyr61 orFisp12. Thymidine incorporation into the trichloroacetic acid-insolublefraction was determined after 24 hour incubation. Logarithmically grownmink lung epithelial cells (Mv1lu, CCL64) were treated with variousconcentrations of TGF-β1 (Gibco-BRL) and 2 μg/ml of Cyr61 or Fisp12 for18 hours; [³H]-thymidine was then added to 1 μCi/ml for 2 hours.Thymidine incorporation was determnined as described above.

Purified recombinant Fisp12 protein did not exhibit any mitogenicactivity under any tested assay conditions. Rather, Fisp12 was able toenhance DNA synthesis induced by fibroblast growth factor in either NIH3T3 fibroblasts or HUVE-cells. This activity was nearlyindistinguishable from that exhibited by Cyr61.

Whereas in fibroblasts and endothelial cells, Cyr61 and Fisp12 enhancegrowth factor-induced DNA synthesis, both proteins can also enhancegrowth factor-mediated actions in another way. It is known that TGF-βacts to inhibit DNA synthesis in epithelial cells (Satterwhite et al.,1994). It was observed that both Cyr61 and Fisp12 enhanced the abilityof TGF-β to inhibit DNA synthesis in mink lung epithelial cells. Thedata demonstrate that both recombinant Cyr61 and Fisp12, purified fromserum-free sources, are not mitogenic by themselves, but have theability to synergize with the actions of polypeptide growth factors.Cyr61 and Fisp12 enhance DNA synthesis induction by FGF, and enhance DNAsynthesis inhibition by TGF-β.

Beyond the use of Cyr61 polypeptides and Fisp12, the present inventioncomprehends the use of CTGF in methods to potentiate the mitogeniceffect of true growth factors, or to screen for true growth factors.Those contemplated uses are in contrast to the reported use of CTGF as amitogen or growth factor itself. U.S. Pat. No. 5.408,040, column 7, line65, to column 11, line 7, incorporated herein by reference hereinabove.It is expected that, in addition to murine and human Cyr61 polypeptides,Fisp12, and CTGF, other ECM signaling molecules, such as members of theCCN family of proteins, will function to potentiate the mitogenicactivity of true growth factors and will not function as true growthfactors themselves.

Further, the invention comprehends methods of screening for modulatorsof mitogen potentiation. A comparative assay exposes subconfluent cellsto an ECM signaling molecule such as Cyr61, a growth factor, and asuspected modulator of an ECM signaling molecule. As a control, similarcells are exposed to the ECM signaling molecule and the growth factor. Afurther control exposes similar cells to the growth factor and thesuspected modulator in the absence of the ECM signaling molecule. Basedon the relative cell proliferation rates, as measured by, e.g.,[³H]-thymidine incorporation, an identification of a suspected modulatoras a promoter of mitogen potentiation (elevated cell proliferation inthe presence of all three molecules) or an inhibitor of mitogenpotentiation (decreased cell proliferation in the presence of the threemolecules) can be made.

Additionally, the invention comprehends the use of ECM signalingmolecules such as Cyr61 polypeptides in methods for treating conditionsor disorders, such as diseases, associated with an under- orover-proliferation of mammalian cells. One of ordinary skill wouldreadily comprehend that Cyr61 polypeptides havinggrowth-factor-enhancing activity are suitable for treating conditionscharacterized by an undesirably low rate of cell proliferation. The useof Cyr61 polypeptides lacking such an activity could be used to treatconditions characterized by an over-proliferation of cells. Any meansknown in the art for delivering the therapeutic polypeptides ormodulators is suitable, including direct injection into a mammal, suchas a human, using any known route (e.g., subcutaneous, intramuscular,intravenous, intraperitoneal), optionally in the presence of a knownadjuvant, excipient, carrier, or vehicle such as a liposome, or by genetherapy using any conventional nucleic acid delivery system. Any ofthese delivery mechanisms may be modified to target delivery of thetherapeutic by mechanisms known in the art (e.g., association with atargeting molecule such as an antibody recognizing a desired cell type).

EXAMPLE 19 Cornea Assay For Angiogenic Factors And Modulators

Another assay for modulators of angiogenesis is an in vivo assay forassessing the effect of a suspected modulator in the presence of an ECMsignaling molecule-related biomaterial, such as Cyr61, on angiogenesisis the Cornea Assay. The Cornea Assay takes advantage of the absence ofblood vessels in the cornea, which in the presence of an angiogenicfactor, results in the detectable development of capillaries extendingfrom the sclera into the cornea. Friedlander et al., Science270:1500-1502 (1995). This ingrowth of new blood vessels from the scleracan be microscopically monitored. Further, the visually determined rateof migration can be used to assess changes in the rate of angiogenesis.These cornea assays may be performed using a wide variety of animalmodels. Preferably, the cornea assays are performed using rats. By wayof example, an assay for suspected modulators of Cyr61 using this assayis disclosed. To perform this assay, Cyr61 is initially titrated usingprimary capillary endothelial cells to determine effectiveconcentrations of Cyr61. Subsequently, Cyr61, in the presence or absenceof a suspected modulator, is surgically implanted into the corneas ofmammalian laboratory animals, e.g., rabbits or rats. In a preferredembodiment, Cyr61 (or Cyr61 and a suspected modulator) is embedded in abiocompatible matrix, using matrix materials and techniques that arestandard in the art. Subsequently, eyes containing implants are visuallyobserved for growth of the readily visible blood vessels within the eye.Control implantations may consist of physiologically balanced buffersembedded in the same type of matrix and implanted into eyes of the sametype of laboratory animal receiving the Cyr61-containing implants.

The development of an in vivo cornea assay for angiogenic factors hasadvantages over existing in vitro assays for these factors. The processof angiogenesis involves four distinct phases: induction of vasculardiscontinuity, endothelial cell movement, endothelial cellproliferation, and three-dimensional restructuring and sprouting. Invitro assays can evaluate only two of these steps: endothelial cellmigration and mitogenesis. Thus, to provide a comprehensive assay forangiogenic factors, an in vivo assay such as the cornea assay ispreferred.

The cornea assay has been used to confirm the effect of angiogenicfactors such as Cyr61, Fisp12, CTGF, and Nov, on the process ofangiogenesis. Moreover, modifying the cornea assay by including any ofthese angiogenic factors and a suspected modulator of their activityresults in a cornea assay for modulators of angiogenesis. For example,in one embodiment of the invention, dose of an angiogenic factor such asCyr61 could be used in cornea assays for positive effectors of theangiogenic activity of Cyr61. An appropriate dose of Cyr61 wouldinitially be determined by titration of the dose response relationshipof Cyr61 with angiogenic events. Inclusion of a control assay lackingCyr61 would eliminate compounds having a direct effect on angiogenesis.In an alternative embodiment of the invention, an effective dose of anangiogenic factor such as Cyr61 could be used to assay for negativemodulators of the activity of an angiogenic factor. In yet anotheralternative embodiment, a corneal implant comprises Cyr61 and anothercorneal implant comprises Cyr61 and a suspected modulator ofangiogenesis. Measurements of the development of blood vessels in theimplanted corneas provides a basis for identifying a suspected modulatoras a promoter of angiogenesis (elevated blood vessel development in thecornea containing an implant comprising the suspected modulator. Arelative decrease in blood vessel development identifies an inhibitor ofangiogenesis.

The rat is preferred as the animal model for the cornea assay.Disclosures in the art have established the rat model as awell-characterized system for analyzing angiogenesis. Parameters such asimplant size, protein release dynamics, and suitable surgicaltechniques, have been well characterized. Although any strain of rat canbe used in the cornea assay, preferred strains will bewell-characterized laboratory strains such as the Sprague-Dawley strain.

Although rats of various sizes can be used in the cornea assay, apreferred size for the rats is 150-200 g/animal. Anesthesia is inducedwith Methoxyflurane and is maintained for 40-60 minutes with sodiumpentobarbital (50 mg/kg, delivered intraperitoneally). The eyes aregently opened and secured in place by clamping the upper eyelid with anon-traumatic hemostat. Two drops of sterile proparacaine hydrochloride(0.5%) are then placed on each eye as to effect local anesthesia. Usinga suitable surgical blade such as a No. 11 Bard Parker blade, anapproximately 1.5 mm incision is made approximately 1 mm from the centerof the cornea. The incision extends into the stromna but not through it.A curved iris spatula approximately 1.5 mm in width and approximately 5mm in length is then inserted under the lip of the incision and gentlyblunt-dissected through the stroma toward the outer canthus of the eye.Slight finger pressure against the globe of the eye helps to steady theeye during dissection. The spatula penetrates the stroma no more thanapproximately 2.5 mm. Once the cornea pocket is made, the spatula isremoved and the distance between the limbus and base of the pocket ismeasured to make sure the separation is at least about 1 mm.

To provide slow release of the protein after implantation in the cornea,protein is mixed with poly-2-hydroxyethylmethacrylate (Hydron®), or anequivalent agent, to form a pellet of approximately 5 μl. Implants madein this way are rehydrated with a drop of sterile lactated Ringerssolution and implanted as described above. After implantation, thecorneal pocket is sealed with erythromycin ointment. After implantation,the protein-Hydron pellet should remain near the limbus of the cornea(cornea-sclera border) and vision should not be significantly impaired.

Following surgery, animals are examined daily for seven days with theaid of a stereomicroscope to check for inflammation and responses. Tofacilitate examination, the animal is anesthetized with Methoxyfluraneand the anesthetic is continuously administered by nose cone duringexamination. During this seven day period, animals are monitored forimplant position and corneal exudate. Animals exhibiting corneal exudateare sacrificed. A preferred method of euthanasia is exsanguination.Animals are initially anesthetized with sodium pentobarbital (50 mg/kg)and then perfused, as described below.

After seven days, animals are perfused with colloidal carbon (e.g.,IndiaInk). Anesthesia is induced with Methoxyflurane, and is maintained withsodium pentobarbital (50 mg/kg, intraperitoneally). Each animal isperfused with 100-200 ml warm (37° C.) lactated Ringers solution per 150g of body mass via the abdominal aorta. Once the snout of the animal iscompletely blanched, 20-25 ml of colloidal carbon is injected in thesame way as the Ringers solution, until the head and thoracic organs arecompletely black. Eyes are then enucleated and fixed. Corneas areexcised, flattened, and photographed.

Each protein is typically tested in three doses, in accordance with thepractice in the art. Those of ordinary skill in the art realize that sixpositive corneal responses per dose are required to support anidentification of an angiogenic response. An exemplary cornea assayincludes three doses of the protein under study, with six rats beingtested at each dose. Additionally, six animals are exposed to abuffer-Hydron implant and serve as negative controls. Exposure of atleast three animals to a known angiogenic factor-Hydron implant serve aspositive controls. Finally, to demonstrate the specificity of anyobserved. response, six animals are exposed to implants containing asingle dose of the protein under study, an excess of neutralizingantibody, and Hydron.

A cornea assay as described above was performed to assess the ability ofCyr61 to induce angiogenesis. Four animals were given negative controlimplants containing a buffer-Hydron pellet (both eyes). Each of theseanimals failed to show any blood vessel development in either eye afterseven days. Six animals received implants containing a biologicallyeffective amount of Fibroblast Growth Factor (0.15 μM) in one eye and acontrol pellet in the other eye; all six showed angiogenic developmentin the eye receiving FGF, none showed neovascularization in the eyereceiving the negative control. Seven animals received 1 μg/ml Cyr61, inone eye and all seven of these eyes showed blood vessel growth; one ofthe seven eyes receiving a negative control showed angiogenicdevelopment. Finally, four animals received implants locally releasing 1μg/ml Cyr61 (Hydron prepared with a 10 μg/ml Cyr61 solution) and aspecific anti-Cyr61 antibody in three-fold excess over Cyr61; none ofthe eyes of this group showed any angiogenic development. Thus, the invivo assay for angiogenesis identifies angiogenic factors such as FGFand Cyr61. The assay also is able to reveal inhibition of angiogenicdevelopment induced ECM signaling molecules such as Cyr61.

EXAMPLE 20 Blood Clotting

ECM signaling molecules are also useful in correcting hemostasis, orabnormal blood clotting. A defect in blood clotting caused by, e.g., lowlevel expression of cyr61 which thereby allows Tissue Factor PathwayInhibitor (TFPI) to act unchecked can be corrected by expression or useof recombinant Cyr61 protein.

Cyr61 can interact with TFPI, a protein that inhibits extrinsic bloodcoagulation. TFPI inhibits blood clotting in a two step process. First,TFPI binds to factor Xa and the TFPI:Xa complex then interacts with theTissue Factor (TF):Factor VIIa complex, thereby inhibiting the lattercomplex. The TF:Factor VIIa complex is the complex that activatesfactors IX and X. By inhibiting TF:VIIa, TFPI reg,ulates coagulation bypreventing the activation of Factors IX and X, required for bloodclotting. The interaction of Cyr61 with TFPI inhibits the activity ofTFPI, thus promoting blood coagulation. Cyr61 is, thus, a tissue factoragonist.

Because of the interaction of Cyr61 and TFPI, Cyr61 can control theability of TFPI to inhibit coagulation, thereby regulating hemostasis. Adefect in Cyr61 may lead to the inability to inhibit TFPI at theappropriate time, resulting in excessive inhibition of tissue factor,thereby preventing clot formation. Deregulated expression of Cyr61 willconversely inhibit the activity of TFPI constitutively, and thus tissuefactor is constantly active, resulting in excessive clotting. When theexpression of cyr61 in endothelial cells is deregulated, one possibleoutcome is thrombosis.

In addition to Cyr61, other ECM signaling molecules, such as Fisp12 andCTGF, have been shown to exert effects on cells participating inangiogenesis. Consequently, it is anticipated that a variety of ECMsignaling molecule-related biomaterials, alone or in combination, may beused in the methods of the invention directed towards modulatinghemostasis.

EXAMPLE 21 Ex vivo Hematopoietic Stem Cell Cultures

To investigate the effect of Cyr61 on the growth of primitivemultipotent stein cells, several assays that distinguish these cellsfrom more mature progenitor cells in a hematopoietic culture areemployed. These assays make use of physicochemical(fibronectin-biniding) or growth and development-related (generation ofprogenitor blast colonies) differences between immature and maturesubsets of cells.

Two cell lines which require conditioned media for growth are used as asource of hematopoietic stem cells (HSC). These cloned, factor-dependentmurine lines are B6Sut (cloned from long term bone marrow culture andcapable of growing in liquid medium without differentiation, butmultipotent in agar, as described in Greenberger et al., Proc. Natl.Acad. Sci. [USA]80:2931 [1983]), and FDCP-mix (cloned from long termbone marrow culture cells infected with the recombinant virussrc-MoMuLV, and are multipotent in agar cultures, as described inSpooncer et al., Nature 310:2288 [1984]). B6Sut cells are propagated inKincaid's medium with 10% fetal calf serum (FCS) and 10% 6×-concentratedWEHI-conditioned medium. Greenberger et al. FDCP-mix cells arepropagated in Fischer's medium with 20% horse serum and 10%6×-concentrated WEHI-conditioned medium. The cell lines are propagatedat 37° C., 5% CO₂.

Various ex vivo or in vitro cultures are assayed for population growthin the presence or absence of exogenously supplied murine Cyr61 orpolyclonal anti-Cyr61 antibodies. Under limiting dilution conditions,the cobblestone area forming cell (CAFC) assay is used to identify cellswith long term repopulating ability. Ploemacher et al., Blood 74:2755(1989); Ploemacher et al., Blood 78:2527 (1991). Cells identified ashaving long term repopulating ability by the CAFC assay are thenanalyzed by measuring three parameters: Rate of population doubling,mitotic index, and rate of DNA synthesis.

Long term cultures, with or without supplementation with Cyr61, areassayed for their levels of primitive HSC in the CAFC assay. van derSluijs et al., Exp. Hematol. 22:1236 (1994). For example, M2-10 B4stromal cells provide a stromal cell layer for either B6Sut or FDCP-mix,which are each subjected to the CAFC assay in the following manner.Stromal cell layers are prepared by inoculating 5×10⁵ M2-10B4stromalcells (a cell line cloned from bone marrow stroma. Sutherland et al.,Blood 78:666 [1991]) into each well of a 96-well culture plate in DMEMwith 10% FCS. When the cells approach confluency, they are rinsed withPBS and irradiated (20 Gy of gamma-irradiation, 1.02-1.04 Gy/minute) toprevent replication of any hematopoietic cells within the stroma,without affecting the stroma's ability to support hematopoiesis.

B6Sut or FDCP-mix cells (sources of HSC) are added to the irradiatedstromal cells in DMEM with 10% FCS, in the presence or absence of Cyr61(10 μg/ml final concentration). After overlaying B6Sut or FDCP-mix onthe stromal cells, the cultures are incubated (e.g., 28-35 days for themurine system) and the number of cobblestone-forming areas (whichidentifies cells with long-term repopulating ability) are counted todetermine the HSC frequency.

In a variation of the above-described CAFC assay, cells (e.g., B6Sut orFDCP-mix) are initially maintained in parallel cultures in the presenceor absence of a Cyr61 polypeptide. Subsequently, CAFC assays areperformed, as generally described above; however, the assay ispreferably performed in the complete absence of CAFC. It is expectedthat cells cultured in the presence of Cyr61 polypeptides will exhibitan increase in the frequency of HSC relative to cells initially culturedin the absence of Cyr61 polypeptides.

Following identification of cells with long-term repopulating ability,population doubling rates are determined, e.g., by microscopicexamination of cell morphology to determine the numbers of long termrepopulating cells (and more mature short term progenitor cells) presentin the various experimental long term cultures. Subsequent investigationof the expansion and differentiation capacities of the potential longterm HSC cultures is used for confirmation of suitable candidate celllines.

The mitotic index is determined according to procedures standard in theart. Keram et al., Cancer Genet. Cytogenet. 55:235 (1991). Harvestedcells are fixed in methanol:acetic acid (3:1, v:v), counted, andresuspended at 10⁶ cells/ml in fixative. Ten microliters of thissuspension is placed on a slide, dried, and treated with Giemsa stain.The cells in metaphase are counted under a light microscope, and themitotic index is calculated by dividing the number of metaphase cells bythe total number of cells on the slide. Statistical analysis ofcomparisons of mitotic indices is performed using the 2-sided pairedt-test.

The rate of DNA synthesis is measured using a thymidine incorporationassay. Various cultures are propagated in 1 μCi/ml [³H]-thymidine (ICNBiomedicals, Inc., Costa Mesa, Calif.) for 24-72 hours. Harvested cellsare then rinsed with PBS and fixed with 10% trichloroacetic acid. DNA isdissolved in 0.1 N NaOH, and thymidine incorporation is determined, forexample by liquid scintillation spectrophotometry.

The function of Cyr61 polypeptides in promoting the maintenance and/orexpansion of long-term hematopoietic stem cell cultures was confirmed byantibody studies. Having determined that Cyr61 is a stroma-associatedcomponent important for the maintenance/expansion of long-term HSCcultures, stromal-contact bone marrow cell cultures were exposed toCyr61 polypeptides in the presence or absence of anti-Cyr61 antibodies.Those cultures in which the Cyr61 activity had been neutralized byexposure to anti-Cyr61 antibodies showed a decrease in vitality of theculture, an increase in the number of visually detectable differentiatedhematopoietic cells in the culture, and a decrease in the cell fractioncomprising HSC, and a concomitant increase in the cell fractioncomprised of committed cells, relative to HSC cultures maintained in thepresence of Cyr61, but the absence of anti-Cyr61 antibodies.Consequently, the activity of Cyr61 polypeptides is expected to beimportant for the stromal-dependent expansion of undifferentiatedhematopoietic stem cells.

The use of an ECM signaling molecule-related biomaterial, such as Cyr61,can be used in the ex vivo expansion of hematopoietic stem cellcultures. In addition, more than one ECM signaling molecule-relatedbiomaterial may be used to expand these cultures. For example, Cyr61,with its expression targeted locally, may be combined with Fisp12, whichis secreted and, consequently, exhibits a more expansive targeting. Asan alternative, CTGF may be substituted for Fisp12, its mouse ortholog.One of skill in the art would be able to devise other combinations ofECM signaling molecule-related biomolecules that are within the spiritof the invention.

Those of ordinary skill in the art will recognize that the successfulexpansion of hematopoietic stem cell cultures in the presence of ECMsignaling molecules such as Cyr61 polypeptides provides a basis for amethod of screening for suspected modulators of that expansion process.As in the other methods of the invention, a suspected modulator iscombined with an ECM signaling molecule such as Cyr61 and exposed toprimitive cells. In parallel, the ECM signaling molecule is exposed tosimilar cells. The relative rates of expansion may be used to identify apromoter, or inhibitor, of the ability of the ECM signaling molecule toexpand pluripotent hematopoietic stem cell cultures.

Cyr61, alone or in combination with other hematopoietic growth factors,may also be used to expand stem cell populations taken from a patientand which may, after expansion, be returned to the patient or othersuitable recipient patient after for example, chemotherapy or othertreatment modalities that result in the depletion of blood cells in apatient. Stem cell populations expanded according to the presentinvention may also be used in bone marrow transplants in a patient inneed thereof. Further, the invention contemplates the delivery of an ECMsignaling molecule such as a Cyr61 polypeptide (including polypeptidefragments that inhibit endogenous Cyr61 activity) to a subject (e.g.,patient) that would benefit from increased, or decreased, HSC productionor hematopoiesis, using delivery means known in the art.

EXAMPLE 22 Organ Regeneration

The role of Cyr61 in the various cellular processes invoked by changesin the cellular growth state indicate that this protein would beeffective in promoting organ regeneration. Towards that end, studieswere conducted to determine the expression profile of murine cyr61 inremaining liver tissue following a partial hepatectomy. (The response ofremaining liver tissue following partial hepatectomy is a model for theliver's response to a variety of injuries, including chemical injuries,e.g., exposure to toxic levels of carbon tetrachloride.)

BALB/c 3T3 (Charles River) mice were subjected to partial hepatectomiesremoving approximately 67% of their liver tissue. Higgins et al., Archs.Path. 12:186-202 (1931). Twenty microgram aliquots of RNA were removedfrom the remaining liver tissue at varying times following the operationand liver RNA was isolated by tissue homogenization followed byguanidinium isothiocyanate, cesium chloride precipitation. Sambrook etal. RNAs were then immobilized on nitrocellulose filters and probed withradiolabeled clones containing various regions of murine cyr61 cDNA.Results were visualized by aultoradiography and indicated that removalof liver tissue induced cyr61 mRNA expression, particularly in cellsfound near the injury site. Consequently, induction of cyr61 expression,e.g., by recombinant techniques, might promote the regeneration oforgans such as liver. For example, cyr61 expression can be controlled,e.g., by introducing recombinant cyr61 constructs that have beenengineered to provide the capacity to control expression of the gene,e.g., by the use of tissue-specific promoters, e.g., the K14 promoterfor expression in skin. The recombinant cyr61 may be introduced to cellsof the relevant organ by gene therapy techniques using vectors thatfacilitate homnologous recombination (e.g., vectors derived fromHerpesviruses. Adenovirus, Adeno-associated Virus, Cytomegalovirus,Baculovirus, retroviruses, Vaccinia Virus, and others). Techniques forintroducing heterologous genes into eukaryotic cells, and techniques forintegrating heterologous genes into host chromosomes by homologousrecombination, are well known in the art.

The development of skin, another organ, is also affected by Cyr61. Theexpression of cyr61 is induced in cells in the vicinity of skininjuries. Also, as described above, Cyr61 has a chemotactic effect(i.e., Cyr61 induces cell migration) on endothelial cells andfibroblasts. Further, Cyr61 induces the proliferation of endothelialcells and fibroblasts. Both processes are involved in the healing ofskin wounds. Accordingly, Cyr61 administration, e.g., by localized ortopical delivery, should promote skin regeneration.

Cyr61 is also highly expressed in lung epithelium. These cells arefrequently injured by exposure to environmental contaminants. Inparticular, lung epithelium is frequently damaged by air-borne oxidants.The administration of Cyr61, e.g., in atomizers or inhalers, maycontribute to the healing of lung epithelium damaged, e.g., byenvironmental contaminants.

EXAMPLE 23 Chondrogenesis—ECM Signaling Molecules Are Expressed inMesenchyme

Some ECM signaling molecules are also expressed in cells, such asmesenchyme cells, that ultimately become a part of the skeletal system.In this Example, Cyr61 is identified as one of the ECM signalingmolecules expressed in mesenchyme cells. Limb mesenchymal cells weregrown in micromass culture as described above on glass coverslips(Fisher) for 3 days. Cultures were fixed in 4% paraformaldehyde in PBS,incubated for 30 minutes at room temperature with 1 mg/ml bovinetesticular hyaluronidase (type IV, Sigma) in 0.1 N sodium acetate (pH5.5) with protease inhibitors phenymethylsulfonyl fluoride (PMSF, 1 mM),pepstatin (1 μg/ml), leupeptin (1 μg/ml), aprotinin (1 μg/ml),aminocaproic acid (50 mM), benzamidine (5 mM), and EDTA (1 mM), blockedwith 10% goat serum in PBS and incubated overnight at 4° C. with primaryantibodies against Cyr61 (Yang et al., [1991]), fibronectin (Gibco) andtenascin (Gibco). Controls were incubated with anti-Cyr61 antibodiesneutralized with 1 μg/ml purified Cyr61. Cultures were subsequentlyincubated with FITC-conjugated goat-anti-rabbit secondary antibody(Zymed), for 1 hour at room temperature.

For whole mount immunohistochemical staining, mouse embryos fromgestational days 10.5 to 12.5 were fixed in 4% paraformaldehyde in PBS,dehydrated in methanol/PBS and stored at −20° C. in absolute methanol.After rehydration, embryos were incubated with anti-Cyr61 antibodies asdescribed in Hogan et al, Development 120:53-60 (1994), incorporatedherein by reference. Controls were incubated with anti-Cyr61 antibodiesneutralized with 1 μg/ml purified Cyr61. Immunostained embryos werefixed, cleared and photographed.

Consistent with the transient expression of the cyr61 mRNA in somiticmesenchymal cells that are differentiating into chondrocytes (O'Brien etal., [1992]), the Cyr61 protein was found in the developing embryonicskeletal system. Cyr61 was localized by whole mount immunohistochemicalstaining to the proximal limb bud mesenchyme in gestational day 10.5 to12.5 embryos. The Cyr61 protein was localized to the developingvertebrae, the calvarial frontal bone and the first brachial arch, aswell as in the heart and umbilical vessels, forming an expressionpattern that was consistent with the cyr61 mRNA expression pattern(O'Brien el al., [1992]).

Cyr61 protein could be detected by immunoblot analysis in whole limbbuds and in micromass cultures of limb bud mesenchymal cells. The levelof Cyr61 protein remained at relatively constant levels throughout the 4day culture period during which chondrogenesis occurred. Usingquantitative immunoblot analysis, Cyr61 was estimated to representapproximately 0.03% of total cellular and extracellular proteins in themesenchymal cell cultures. Cyr61, tenascin (Gibco), and fibronectin werelocalized to the cartilage nodules by immunofluorescent staining in themesenchymal cell cultures. Cyr61 and tenascin were primarily localizedamong the intranodular cells, while a fibrillar staining pattern wasalso observed around and between the cartilage nodules withanti-fibronectin antibodies. A similar immunofluorescent stainingpattern was observed in transverse sections of the micromass culturesfor all three antibodies. Together, these results show that endogenousCyr61 is localized in the developing limb bud mesenchyme, both in vivoand in vivo.

EXAMPLE 24 Chondrogenesis—ECM Signaling Molecules Promote Cell Adhesion

Cyr61 is a secreted protein that mediates the adhesion of fibroblastsand endothelial cells to non-tissue culture-treated plastic surfaces(Kireeva et al., [1996]). The attachment of limb bud mesenchymal cellson non-tissue culture dishes coated with BSA, Cyr61, tenascin, andfibronectin, were compared.

Cyr61, fibronectin (Gibco), or tenascin (Gibco) were diluted in 0.1%protease-free bovine serum albumin (BSA) in PBS with 0.5 mM PMSF, to afinal concentrations of 10 or 50 μg/ml. A 10 μl drop/well was placed ina non-tissue culture treated 24-well plate (Corning), and incubated atroom temperature for 2 hours. The wells were blocked with 1% BSA in PBSfor 1 hour at room temperature, and rinsed with serum-free MEM (ModifiedEagle's Medium). Limb mesenchymal cells, suspended at 5×10⁵ cell/ml inserum-free MEM, were added at a volume of 400 μl/well, and incubated at37° C., 5% CO₂ for 1 or 3 hours. At each time point, the cell suspensionwas removed, the wells were rinsed with MEM and the remaining adherentcells were photographed.

Cells attached poorly to BSA-coated dishes, but adhered as clusters ofrounded cells to Cyr61- and tenascin-coated dishes within 1 hour ofplating. In contrast, cells plated on fibronectin-coated dishes attacheduniformly and started to spread. When cells were allowed to attach for 3hours, many more adherent cells were observed. Furthermore,intercellular clustering and rounded cell morphology were maintained incells plated on Cyr61 and tenascin, while cells plated on fibronectinspread to form a monolayer. These observations show that Cyr61 mediatesthe adhesion and maintenance of a rounded cellular morphology which isconducive for mesenchymal cell chondrogenesis (Zanetti et al., Dev.Biol. 139:383-395 [1990]; Solursh et al., Dev. Biol. 94:259-264 [1982]),similar to that previously reported for tenascin (Mackie et al., J. CellBiol. 105:2569-2579 [1987]).

As mentioned previously, ECM signaling molecules such as Cyr61 may beused in methods of screening for modulators of cell adhesion, including,but not limited to, the adhesion of chondrocytes. The comparative assay,described above, measures the relative adhesion levels of cells exposedto a combination of an ECM signaling molecule and a suspected modulatorof cell adhesion and cells exposed to the ECM signaling molecule alone,whereby the relative levels provide a basis for identifying either apromoter or an inhibitor of cell adhesion.

EXAMPLE 25 Chondrogenesis—ECM Signaling Molecules Promote CellAggregation

Since aggregation is an essential step for chondrogenic differentiation(Solursh, M., In The role of extracellular matrix in development, pp.277-303 (Trelstad, R., ed.) (Alan R. Liss, New York 1984)), the abilityof Cyr61 to

EXAMPLE 26 Chondrogenesis—ECM Signaling Molecules Promote CellProliferation

Some ECM signaling molecules, such as Cyr61, affect chondrogenesis, asrevealed by effects on limb bud mesenchyme cells in micromass culture,as described above. Ahrens et al., Dev. Biol. 60:69-82 (1977), hasreported that these cells, in micromass culture, undergo chondrogenesisin a manner similar to the in vivo process. Mesenchyme cells wereobtained from mouse embryonic limb buds by trypsin digestion (1 mg/ml,1:250 dilution of porcine pancreatic trypsin, Sigma Chemical Co.). Cellswere explanted in plastic tissue culture wells and allowed to attach for2 hours at 37° C., 5% CO₂. Cells were then incubated for 24 hours at 37°C., 5% CO₂ in MEM with 10% FBS, penicillin (50 U/ml), and streptomycin(50 μg/ml). At this point, the composition of the medium was changed bysubstituting 4% NuSerum (Collaborative Biomedical Products, Inc.) for10% FBS. Individual cultures then received Cyr61, fibronectin, heparin(each at approximately 1 μg/ml) or buffer as a negative control. Anadditional control was provided by adding a 1:100 dilution ofaffinity-purified anti-Cyr61 antibody (approximately 13 μg/ml stocksolution), elicited and purified by standard techniques. Harlow et al.

Cell proliferation was assessed by the thymidine assay, described above,and by microscopic cell counts. Chondrogenesis was assessed byquantifying the incorporation of [³⁵S]-sulfate (ICN Biomedicals, Inc.)into sulfated glycosaminoglycans, and by qualitatively determnining theextent of chondrogenesis by cell staining with Alcian Blue. Cultures,described above, were labeled with 2.5 μCi/ml [³⁵S]-sulfate for 18hours, washed twice in PBS, fixed with Kahle's fixative (Pepper et al.,J. Cell Sci. 109:73-83 [1995]) and stained for 18 hours in 0.5% AlcianBlue, pH 1.0. The extent of chondrogenesis is correlated with theintensity of Alcian Blue staining. San Antonio et at., Dev. Biol.115:313-324 (1986). The results show that Cyr61 specifically increasedlimb bud mesenchyme cell proliferation and aggregation, leading toenhanced chondrogenesis.

In addition to demonstrating that purified Cyr61 enhanced growthfactor-induced DNA synthesis in fibroblasts and endothelial cells, theeffects of Cyr61 on cell proliferation were directly examined. Cellproliferation during the 4 day culture period was determined by countingcell number and by incorporation of [³H]-thymidine. To determine cellnumber, cells were harvested by trypsin/EDTA (Sigma) and counted with aCoulter counter. In parallel cultures, [³H]-thymidine (1 μCi/ml; ICN)was added to the media for 18 hours and incorporation in theTCA-insoluble layer was determined by liquid scintillation counting.Purified Cyr61 protein added to limb mesenchymal cells both increasedcell number and enhanced DNA synthesis after 2 and 3 days in culture,although the total cell number in Cyr61-treated and Cyr61-untreatedcultures leveled off at the same level after 4 days.

The role of Cyr61 in chondrogenesis may also improve the integration ofprosthetic devices. For example, skeletal injuries and conditionsfrequently are treated by the introduction of a prosthesis e.g., hipprosthesis, knee prosthesis. Beyond questions of histocompatibility, thesuccessful implantation of a prosthetic device requires that the foreignelement become integrated into the organism's skeletal structure. Thecapacity of Cyr61 polypeptides to affect cell adhesion, migration, andproliferation, and the ability of Cyr61 polypeptides to induce thedifferentiation of mesenchyme cells into chondrocytes, should provevaluable in the treatment of skeletal disorders by prosthesisimplantation. For example, integration of a prosthetic device bychondrocyte colonization would be promoted by therapeutic treatmentsinvolving the administration of Cyr61 in a pharmaceutically acceptableadjuvant, carrier or diluent, using any of the administration routesknown in the art or by coating the prosthesis device with Cyr61polypeptides in a suitable carrier. The carrier may also be aslow-release type vehicle to allow sustained release of thepolypeptides.

As noted in previously, the methods of the invention include a method ofscreening for modulators of cell proliferation, including chondrocytes.A comparison of the relative rates of cell proliferation in the presenceof a control comprising an ECM signaling molecule alone (e.g. Cyr61) andin the presence of a combination of an ECM signaling molecule and asuspected modulator of cell proliferation provides a basis foridentifying a suspected modulator as a promoter, or inhibitor, ofchondrocyte proliferation.

EXAMPLE 27 Chondrogenesis—ECM Signaling Molecules Promote Chondrogenesis

Chondrogenic differentiation was quantitated by incorporation of[³S]-sulfate (ICN) into sulfated glycosaminoglycans and assessedqualitatively by Alcian Blue staining. Cultures were radiolabeled with2.5 μCi/ml [³⁵S]-sulfate for 18 hr, fixed with Kahle's fixative andstained with 0.5% Alcian Blue, pH 1.0 (Lev et al., 1964). The extent ofchondrogenesis is correlated with the intensity of Alcian Blue staining(San Antonio et al., 1986). [³⁵S]-Sulfate incorporation in the fixedcell layer was quanititated by liquid scintillation counting.

Exogenous purified Cyr61 protein promoted limb mesenchymal cellaggregation and resulted in greater Alcian blue-staining cartilaginousregions in micromass cultures, suggestive of a chondrogenesis-promotingeffect. This effect was quantified by the incorporation of [³⁵S]-sulfateinto sulfated glycosaminoglycans (San Antonio et al., 1986) inCyr61-treated micromass cultures. Exogenous Cyr61 enhanced [³⁵S]-sulfateincorporation in a dose-dependent manner, resulting in a 1.5-fold and3.5-fold increase with 0.3 and 5 μg/ml Cyr61, respectively, and wascorrelated qualitatively by increased Alcian Blue staining. The increaseobserved at the 5 μg/ml Cyr61 dose (120 nM) is an under-estimation ofthe actual extent of chondrogenesis, since some of the large cartilagenodules which were formed at this dose detached from the dish. Culturestreated with 10 μg/ml Cyr61 formed a more massive mound of cartilage.

A review of the literature indicated that chondrogenesis in limbmesenchymal cell micromass cultures was increased 2-fold with theaddition of 10 μg/ml heparin (San Antonio et al., [1987]; Resh et al.,[1985]) and 3-fold with 50 μg/ml tenascin (200 nM) (Mackie et al.,[1987]). The results demonstrated Cyr61-treated cultures still showed anapproximately 2-fold increase in normalized sulfate incorporation overcontrol, indicating that Cyr61 promotes a net increase inchondrogenesis. On culture day 2, the sulfate/cell number ratio waslower in Cyr61-treated cultures compared to controls and is reflectiveof a low level of [³⁵S]-sulfate incorporation relative to cell number,since mesenchymal cells are mostly proliferating rather thandifferentiating in these early stage cultures (Ede, 1983).

The presence of endogenous Cyr61 in these cells, both in vivo and invitro, indicates that Cyr61 may indeed function biologically to regulatechondrogenic differentiation. The ability of exogenously added purifiedCyr61 to promote intercellular aggregation and to increase [³⁵S]-sulfateincorporation and Alcian-blue staining in limb mesenchymal cellsdemonstrates that Cyr61 can act as a chondrogenesis enhancing factor. Asshown above in Example 11, anti-Cyr61 antibodies can neutlralize boththe cell adhesion and DNA-synthesis enhancement activities of Cyr61.Anti-Cyr61 antibodies were added to the mesenchymal cell culture mediaor mixed the cell suspension prior to plating. Chondrogenesis wasinhibited in the cultures treated with anti-Cyr61 antibodies. asdemonstrated by decreases of [³⁵S]-sulfate incorporation to 50% and 30%of controls, when antibodies were added to the media, and mixed with thecells, respectively. These observations were correlated with decreasedAlcian Blue staining. However, mixing of the anti-Cyr61 antibodies withmesenchymal cells prior to plating resulted in complete detachment insome of the treated cultures within 24 hours.

To eliminate the possibility of an unidentified component in theantibody preparation as a cause of cell detachment, anti-Cyr61 antibodywas preincubated with 1 μg/ml purified Cyr61 protein prior to mixingwith cells. The inhibition of chondrogenesis in mesenchymal cells mixedwith neutralized anti-Cyr61 antibodies was abolished.

Generally, the invention contemplates a method of screening formodulators of chondrogenesis. A comparative assay involves the exposureof in such a way that the ECM signaling molecule's activities aremodulated or abolished.

Furthermore, because of the role played by ECM signaling molecules suchas Cyr61 in regulating chondrogenesis (i.e., skeletal development),genetic manipulations that alter the expression of human Cyr61 may provemedically important for prenatal screening methods and gene therapytreatments related to skeletal conditions, in addition to angiogenicconditions. For example, the cyr61 gene is expressed when mesenchymalcells of both ectodermal and mesodermal origins differentiate to formchondrocytes. Thus, one of the roles that Cyr61 might play is toregulate the commitment of mesenchyme cells to chondrocyte cell lineagesinvolved in skeletal development. Consistent with this view, transgenicmice overexpressing cyr61 ectopically are born with skeletalabnormalities. In all cases examined, the presence of the skeletaldeformities correlates with expression of the transgene. These resultssuggest that the human form of Cyr61 may also regulate chondrogenesisand skeletal development. It is also possible that the human cyr61 genemay correspond to a genetic locus already known to affect skeletaldevelopment or birth defects relating to bone morphogenesis. Knowledgeof the human Cyr61 protein sequence, presented in SEQ ID NO:4 herein,and the coding sequence of the cDNA, presented in SEQ ID NO:3 herein,provide the basis for the design of a variety of gene therapyapproaches.

This information also provides a basis for the design of probes usefulin genotypic analyses, e.g., Restriction Fragment Length Polymorphismanalyses. Such analyses are useful in the fields of genetic counseling,e.g., in diagnosing diseases and conditions and the likelihood of theiroccurrence, as well as in forensic analyses.

By way of example, the materials of the present invention are useful inthe prenatal screening for a variety of conditions or disorders,including blood disorders, skeletal abnormalities, and cancerousconditions. Well known techniques for obtaining fetal cells, e.g.,amniocentesis, provide the materials α₆β₁ for both Cyr61 and CTGF. Inaddition to its involvement in fibroblast adhesion, Cyr61 inducesangiogenic factors such as vascular endothelial growth factor (i.e.,VEGF) in these cells. It is expected that CTGF will also induce VEGFexpression.

Both Cyr61 and CTGF serve as bona fide signaling molecules actingthrough adhesion receptors. Fibroblast adhesion to Cyr61 and CTGFresults in focal adhesion plaques, which can be visualized by stainingwith either anti-β₁ antibodies, or anti-focal adhesion kinase (FAK),anti-paxillin, or anti-vinculin antibodies. Morphologically, fibroblastsadhered to Cyr61 form filipodia, consistent with a migratory response.Further, adhesion results in the rapid tyrosine phosphorylation of FAKand paxillin, as well as activation of MAP kinase and JNK kinase throughphosphorylation. Adhesion to Cyr61 results in altered gene expression infibroblasts, such as induction of MMP1 (collagenase, matrixmetalloproteinase 1).

The α₆β₁ integrin is the adhesion receptor for Cyr61 and CTGF infibroblasts. Moreover, Cyr61 mediated adhesion through α₆β₁ involvesconcurrent binding to heparan sulfate proteoglycan. By contrast, Cyr61adhesion through α_(V)β₃ in endothelial cells does not require bindingto proteoglycans. Thus, the adhesion of endothelial cells consists oftwo components: one mediated through integrin α_(V)β₃ and the othermediated through α₆β₁. A small amount of Cyr61-mediated adhesion inendothelial cells that could not be blocked by LM609 has been observed.This residual adhesion was completely blocked by anti-α₆β₁ antibodies.Thus α₆β₁ is the primary receptor for Cyr61 in fibroblasts, and thesecondary receptor in endothelial cells.

The functional-blocking monoclonal antibodies against integrins werepurchased from Chemicon Inc: JBIA (anti-β₁); FB12 (anti-α₁); PIE6(anti-α₂): PIB5 (anti-α₃); PIH4 (anti-α₄); PID6 (anti-α₅); NKI-GoH3(anti-α₆); P3G8 (anti-α_(V)); LM609 (anti-α_(V)β₃). Polyclonalanti-Cyr61 antibody was raised in rabbits and affinity purified asdescribed (Kireeva et al., [1997]). Synthetic peptides GRGDSP (SEQ IDNO:31) and GRGESP (SEQ ID NO:32) were purchased from Gibco-BRL. Heparin,Chondroitin sulfate A, Chondroitin sulfate C, and Decorin were fromSigma. Chondroitin sulfate B (Dermatan sulfate) and low molecular weight(3K) heparin were from Fluka.

To further characterize Cyr61-mediated fibroblast adhesion, Cyr61variants or mutants were constructed that harbored alanine substitutionswithin the putative heparin-binding sites between, approximately, aminoacids 280-290 and amino acids 306-312. Constructions involvedsite-directed mutagenesis using a two-step polymerase chain reaction(PCR) procedure. Briefly, two overlapping internal oligonucleotideprimers containing the altered sequences in opposite orientation alongwith outside primers were used in two separate PCR reactions. Mousecyr61 cDNA was used as a template in the first PCR reaction. Resultingproducts were gel purified, combined, and used as a template for thesecond PCR reaction. The final mutant PCR product was digested withBsrGI, which cuts at sites flanking the mutated sequences. The BsrGIfragment containing the wild-type cyr61 cDNA in the pSG5 vector wassubstituted with the mutated PCR product. The orientation and mutationswere confirmed by DNA sequencing. Finally, the mutant cyr61 cDNA wasreleased from pSG5 by digestion with EcoRI from pSG5 and cloned into theBaculovirus expression vector pBlueBac 4.5 (Invitrogen).

For the H1 mutant, the internal primers were5′-GCGGCATGCAGCGCGACCGCGAAATCCCCAGAACCAGTC-3′ (primer fH1; SEQ ID NO:18)and 5′-TCGCGCTGCATGCCGCGCCCGC-TTTTAGGCTGCTGTACACTG-3′ (primer rH1; SEQID NO:19). For the H2 mutant, the internal primers were5′-GTCGCGGCATACGCGCCCAAATAC-TGCGGCTC-3′ (primer fH2; SEQ ID NO:20) and5′-GCGCGTATGCCGC-GACACTGGAGCATCCTGC-3′ (primer rH2; SEQ ID NO:21). Theoutside primers used in the second PCR reaction for each mutant were5′-CAGACCACGTCTTGGTCC-3′ (upstream PCR primer; SEQ ID NO:22) and5′-GAATAGGCTGTACACTCGG-3′ (downstream PCR primer; SEQ ID NO:23). Toconstruct a double mutant (dmcyr61), the H2 mutant cyr61-containingpretreating cells with heparinase I (2 U/ml, Sigma) or chondroitinaseABC (2 U/ml, Sigma) at 37° C. for 30 minutes.

The ability of Cyr61 to mediate cell adhesion in normal fibroblasts wasinvestigated. Microtiter wells were coated with purified recombinantCyr61 protein, and 1064SK primary human foreskin fibroblasts wereallowed to adhere under serum-free conditions. In particular, washed1064SK fibroblasts were detached with 2.5 mM EDTA and resuspended inserum-free DMEM medium at 2.5×10⁵ cells/ml. 50 μl cell suspensions wereplated on microtiter wells coated with varying concentrations (0.5-5.0μg/ml) of Cyr61 protein. After incubation at 37° C. for 30 minutes,adherent cells were fixed and stained with methylene blue. Extracted dyewas quantified by absorbance at 620 nm and means from three trialsshowed an absorbance of about 0.3 A₆₂₀ (0.5 μg/ml Cyr61), with theabsorbance ranging from 0.45-0.55 A₆₂₀ for Cyr61 solutions of 1-5 μg/ml,respectively. Adhesion of 1064SK cells to Cyr61 was dose-dependent andsaturable.

In another experiment, microtiter wells were coated with either BSA,Cyr61 (2 μg/ml), or vitronectin (VN; 0.5 μg/ml) and blocked withaffinity-purified anti-Cyr61 antibodies for one hour at 37° C. before1064SK cells were plated. The A₆₂₀ for BSA-coated wells was about 0.05with or without anti-Cyr61 blocking antibody. With Cyr61-coated wells,the A₆₂₀ was 0.45 (without blocking antibody) or 0.15 (with blockingantibody). With VN-coated wells, the A₆₂₀ was about 0.55, with orwithout blocking antibody. These data are means of triplicatedeterminations. The results showed that affinity-purified anti-Cyr61antibodies inhibited 1064SK cell adhesion to Cyr61 but not tovitronectin, indicating that the ability to mediate fibroblast adhesionwas an intrinsic property of the Cyr61 protein.

The effects of divalent cations in cell adhesion to Cyr61 was alsoinvestigated. Cells were added to microtiter wells coated with Cyr61 (2μg/ml), Type I collagen (Col. 1, 2 μg/ml), Vitronectin (VN, 0.5 μg/ml)or BSA (control); EDTA (2.5 m) or Mg²⁺ (5.0 mM) was added to some cells.Cells were plated on microtiter wells coated with Cyr61 (2 μg/ml), andone of the following components was added: Nothing, Ca²⁺, Mg²⁺,Ca²⁺/Mg²⁺, or Ca²⁺/Mn²⁺(5.0 mM each); the A₆₂₀ values were 0.07, 0.46,0.07, 0.46, 0.50, 0.08, and 0.48, respectively. Data are means fromtriplicate determinations. Fibroblast adhesion to Cyr61 was completelyblocked by 2.5 mM EDTA (about 0.12 A₆₂₀), and was restored by theaddition of 5.0 mM Mg²⁺(0.50 A₆₂₀). As expected, similar effects wereobserved for fibroblasts plated on collagen (0.10 A₆₂₀ with EDTA, 0.58A₆₂₀ with Mg²⁺) or vitronectin (0.05 A₆₂₀ with EDTA, 0.55 A₆₂₀ withMg²⁺). The presence of Ca²⁺ abolished cell adhesion to Cyr61 competely,whereas the addition of Mg²⁺ or Mn²⁺ had no effect. Inhibition by Ca²⁺is characteristic of adhesion through some members of the β₁ family ofintegrins, including receptors that bind type I collagen, laminin, andvitronectin (integrins α₂β₁, α₆β₁, and α_(V)β₁) in fibroblasts, as wellas the lymphocyte-specific integrin α_(L)β₂. This observation alsohelped to exclude some integrins whose adhesive properties are supportedby Ca²⁺. The presence of Mn²⁺, but not Mg²⁺, was able to overcome theinhibitory effect of Ca²⁺ on cell adhesion to Cyr61, suggesting thatMn²⁺ can bind the Cyr61 adhesion receptor with higher affinity thanCa²⁺.

A number of integrins expressed in fibroblasts, notably the α_(V)integrins (α_(V)β₁, α_(V)β₃ and α_(V)β₃ vitronectin receptors) andintegrin α₅β₁ (fibronectin receptor), are sensitive to inhibition byRGD-containing peptides. The effects of RGD peptides on Cyr61-mediatedfibroblast binding was tested by plating cells in wells coated withCyr61 (2 μg/ml). Type I collagen (2 μg/ml) or vitronectin (0.5 μg/ml), 2mM GRGDSP or GRGESP peptide was then added. For Cyr61-coated wells, noaddition, GRGDSP, or GRGESP gave A₆₂₀ values of about 0.50, 0.48, and0.46, respectively. For Type I collagen, the A₆₂₀ values were 0.50, 0.47and 0.49, respectively, and for VN, the values were 0.45, 0.05, and0.41, repectively.

Cells were also pre-incubated with 40 μg/ml monoclonal antibodiesagainst the integrin α₅ or the integrin α_(V) subunits at roomtemperature for 30 minutes, then plated on wells coated with Cyr61 (2μg/ml), vitronectin (0.5 μg/ml) or fibronectin (FN, 1 μg/ml). ForCyr61-coated wells, the A₆₂₀ values were 0.75 (no addition), 0.73(anti-α_(V)), and 0.77 (anti-α₅); the corresponding values for VN were0.78, 0.32, and 0.72, for fibronectin, the corresponding values were0.72, 0.65, and 0.36. The data are means, representative of threeexperiments. The peptide RGDSP, but not the control peptide RGESP,completely abolished 1064SK cell adhesion to vitronectin. By contrast,RGDSP had no effect on fibroblast adhesion to Cyr61 or type I collagen.This result indicated that fibroblast adhesion to Cyr61 is not mediatedthrough either the α_(V) integrins (α_(V)β₁ α_(V)β₃, and α_(V)β₅) orα₅β₁.

1064SK cells were also challenged by pre-incubation with monoclonalantibodies specifically recognizing the α_(V) and α₅ integrin subunits.Whereas 1064SK adhesion to vitronectin and fibronectin was blocked bymonoclonal antibodies against α_(V) and α₅, respectively, theseantibodies had no effect on adhesion to Cyr61. Thus, 1064SK fibroblastadhesion to Cyr61 is mediated through one of the subset of β₁ integrins(e.g., α₂β₁, α₃β₁, or α₆β₁) known to be inhibited by Ca²⁺ andinsensitive to inhibition by RGD-containing peptides.

The 1064 SK cells were also analyzed using the monoclonal antibody JBIA,which binds the β₁ integrin subunit. Cells were pre-incubated with theantibody (50 μg/ml) before plating on microtiter wells coated with Cyr61(2 μg/ml), Type I collagen (2 μg/ml), or vitronectin (0.5 μg/ml).Anti-β₁ antibody inhibited cell adhesion to Cyr61 (A₆₂₀ of 0.53 withoutantibody and 0.14 with antibody) by 75%, confirming that 1064SKfibroblast adhesion to Cyr61 requires the involvement of a β₁ integrin.As expected, adhesion to type I collagen was inhibited by about 62%(A₆₂₀ of 0.56 without and 0.21 with antibody) by the anti-β₁ antibody,whereas adhesion to vitronectin was unaffected (A₆₂₀ of 0.48 without,and 0.44 with, antibody). Data are means, representative of threeexperiments.

To identify the specific β₁ integrin that mediates fibroblast adhesionto Cyr61, the inhibitory effects of function-blocking monoclonalantibodies were tested against various integrin α subunits. Cells werepre-incubated with monoclonal antibodies against the integrin β1 subunit(50 μg/ml, see results described above) or the integrin α₆ subunit (20μg/ml) at room temperature for 30 minutes, then plated on wells coatedwith Cyr61 (2 μg/ml), laminin (5 μg/ml), or fibronectin (1 μg/ml). Thedata are means, representative of at least three experiments. Monoclonalanti-α₆ antibody blocked 1064SK fibroblast adhesion to Cyr61 (A₆₂₀ of0.53 without, and 0.08 with, antibody) by more than 80%, while having noeffect on adhesion to fibronectin. Adhesion to laminin, a ligand forintegrin α₆β₁, was only partially blocked (A₆₂₀ of 0.62 without, and0.48 with, antibody, or a block of about 22%). Cells were pre-incubatedwith 40 μg/ml monoclonal antibodies against integrin α1, α2, α3 or α4,or treated with a cocktail of α1, α2 and α3 antibodies (40 μg/ml each)at room temperature for 30 minutes, then plated on wells coated withCyr61 (2 μg/ml), vitronectin (0.5 μg/ml), or Type I collagen (2 μg/ml).Data are means, representative of three experiments, and are presentedin Table II.

TABLE II Antibody Cyr61 (A₆₂₀) VN (A₆₂₀) Coll. I (A₆₂₀) None (control)0.58 0.59 0.74 anti-α₁ 0.54 0.57 0.62 anti-α₂ 0.60 0.58 0.31 anti-α₃0.55 0.54 0.63 anti-α₄ 0.56 0.55 0.72 anti-α₁ + anti-α₂ + anti-α₃ 0.560.56 0.10Function-blocking monoclonal antibodies against integrin α₁, α₂, α₃ orα₅ subunits, or a combination of antibodies against α₁, α₂ and α₃ hadlittle effect on Cyr61-mediated cell adhesion (Table II). In contrast, amixture of anti-α₁, anti-α₂, and anti-α₃ antibodies almost completelyinhibited fibroblast adhesion to collagen. Thus, 1064SK fibroblastadhesion to Cyr61 is mediated through integrin α₆β₁.

The role of heparin in the binding of fibroblasts to Cyr61 was alsoexamined. First, various amounts of soluble heparin were added tosuspensions of 1064SK fibroblasts prior to plating on either Cyr61- orfibronectin-coated wells. In particular, cells were plated on wellscoated with Cyr61 (2 μg/ml) or fibronectin (1 μg/ml). Different amountsof heparin (0.001-1000 μg/ml) were included in the cell suspensionbefore plating. Cells were plated on wells coated with Cyr61 (2 μg/ml)or vitronectin (0.5 μg/ml). Data in trms of means, representative of atleast three experiments, showed that heparin levels as low as 1 ng/mldetectably inhibited binding, with 0.1 μg/ml or more completelyinhibiting cell adhesion to Cyr61. However, heparin levels up to 1000μg/ml had no effect on cell adhesion to fibronection.

The influence of chondroitin sulfates on cell adhesion to Cyr61 wasadditionally examined. Cells were plated on wells coated with Cyr61 (2μg/ml) or vitronectin (0.5 μg/ml). Chondroitin sulfate A (1 mg/ml),chondroitin sulfate B (100 μg/ml), chondroitin sulfate C (10 mg/ml), ordecorin (100 μg/ml) was included in the cell suspension before plating.The data (three trials) showed that 1 mg/ml chondroitin A or 10 μg/ml ofeither chondroitin B or decorin inhibited cell adheshion to Cyr61,chondroitin C failed to inhibit adhesion to Cyr61 at all concentrationstested (0.01-1000 μg/ml). However, the concentrations of chondroitinsulfates A, B, or decorin needed for this inhibition were orders ofmagnitude higher than the effective inhibitory concentration of heparansulfate (e.g., 0.1 μg/ml heparan sulfate versus 1 mg/ml chondroitinsulfate A).

The addition of sodium chlorate (an inhibitor of proteoglycan sulfation)to cell suspensions in Cyr61-treated (2 μg/ml) wells resulted in adose-dependent response, with 90% inhibition of adhesion at 40 mM sodiumchlorate. In contrast, this concentration of chlorate resulted in only a10-20% inhibition of cell adhesion to other substrates (fibronectin,type I collagen, vitronectin, and laminin). Data are means,representative of three experiments. The chlorate inhibition study wasconducted by culturing cells in media containing 0-50 mM sodium chloratefor 24 hours, washing, and harvesting as described above, then platingon Cyr61 92 μg/ml), fibronectin (1 μg/ml), Type I collagen (Coll. 1, 2μg/ml), vitronectin (VN, 0.5 μg/ml), or laminin (5 μg/ml).Chlorate-mediated adhesion inhibition was resuced by the addition of 10mM MgSO₄. Cells were cultured in media containing 50 mM sodium chlorate,or 50 mM sodium chlorate plus 10 mM magnesium sulfate for 24 hours,washed and harvested, then plated on wells coated with Cyr61 (2 μg/ml),vitronectin (0.5 μg/ml), or fibronectin (1 μg/ml).

The 1064SK fibroblasts were also treated with heparatinase, whichrendered cells unable to adhere to Cyr61 (A₆₂₀ of 0.13 with, and 0.59without, treatment), but had no effect on cell adhesion to vitronectin(A₆₂₀ of 0.63 with, and 0.70 without, treatment) or fibronectin (A₆₂₀ of0.66 with, and 0.74 without, treatment). In contrast, chondroitinase A,B, C had no effect (A₆₂₀ values of 0.57, 0.69, and 0.65, for Cyr61,vitronectin and fibronectin, respectively) on cell adhesion, indicatingthat chondroitin sulfates do not contribute significantly to humanforeskin fibroblast adhesion to Cyr61. Thus, cell surface proteoglycans,such as heparan sulfate proteoglycans, are involved in Cyr61-mediatedfibroblast cell adhesion. The adhesion of human fibroblasts to Cyr61 ismediated through integrin α₆β₁, and sulfated proteoglycans play a rolein that adhesion.

Mutant Cyr61 proteins deficient in heparin binding were generated toexamine the effect of such changes on fibroblast adhesion. Conventionalsite-directed multagenesis techniques were used to produce mutant Cyr61polypeptides having altered heparin-binding motifs. Two pulativeheparin-binding motifs were found within the carboxyl-terminal domain inCyr61 that conform to the consensus XBBXB sequence for heparin binding(where B demots basic amino acid residues such as lysine or arginine).Site-directed mutagenesis was used to replace the lysine and arginineresidues in the motifs with alanine, thus creating two Cyr61 variants(H1 and H2) each having one of the two heparin binding motifs mutated.In addition, both motifs were mutated in a Cyr61 double mutant (DM)variant. A comparison of the mutated amino acid sequenceH₂NSLKAGAACSATAKSPEPVRFTYAGCSSVAAYAPKYCG—CO₂H (SEQ ID NO:30) withresidues 278-314 of SEQ ID NO:2 (wild-type mouse Cyr61), shows clustersof amino acid changes between residues 280-290 (H1, underscored above)and between residues 305-310 (H2, underscored above); both sets ofclustered changes are found in DM. These mutations were created usingthe full-length cyr61: the mutant constructs were expressed in, andpurified from, recombinant Baculovirus-transformed insect cells. Equalamounts of conditioned media of insect SF9 cells infected withBaculovirus expressing wild-type or mutant Cyr61 protein were loaded onCL-6B Heparin Sepharose columns. After washing with 20 bed volumes ofRIPA buffer, bound protein was eluted with RIPA buffer containingincreasing concentrations of sodium chloride. Equal amounts of eluatefrom each fraction were analyzed on SDS-PAGE gels followed by Westernblotting to visualize Cyr61 protein. Antibodies used were rabbitpolyclonal antibodies against bacterial GST-Cyr61. The H1 mutant Cyr61polypeptide eluted over the range of 04-0.8 M NaCl; DM eluted during thewashing and up to 0.25 M NaCl; and wild-type Cyr61 eluted at 0.8-1.0 MNaCl. These elution profiles indicate that H1 and H2 exhibited somewhatdecreased heparin-binding affinities, whereas DM was severely deficientin heparin binding.

To examine the activities of these Cyr61 variants (i.e., mutant Cyr61proteins), various concentrations of the variants were separately coatedonto microtiter wells and 1064SK fibroblasts were added. Adhesion assayswere performed by optionally pre-incubating cells with 20 μg/mlmonoclonal anti-α₆ antibody at room temperature for 30 minutes, thenplating on wild-type Cyr61, mutant Cyr61 H1, mutant Cyr61 H2, orvitronectin (0.5 μg/ml). The results (three trials) showed that both H1(e.g., 2.5 μg/ml) and H2 (e.g., 2.5 μg/ml) were able to supportfibroblast adhesion with adhesion isotherms that were comparable to thatof wild-type Cyr61 (e.g., 2 μg/ml), although maximal adhesion wasreached at a lower concentration of wild-type protein (1 μg/ml) comparedto the mutant proteins (2.5-5.0 μg/ml). Moreover, adhesion to either H1or H2 was blocked by antibodies against the integrin subunit α₆,indicating that cell adhesion to the mutant Cyr61 proteins is alsomediated through integrin α₆β₁. In particular, the antibody inhibitedCyr61-mediated binding by 78%, H1-mediated binding by 69%, andH2-mediated binding by 70%, fibroblast binding to vitronectin was onlyinhibited by 9%. Thus, the integrin α₆β₁ binding sites of Cyr61 aredistinct from the heparin-binding sites mutated in H1 and H2. MutantCyr61 proteins that preserved wither heparin-binding site exhibitedsufficient residual heparin-binding activity to support fibroblastadhesion. In contrast, DM was unable to support 1064SK fibroblastadhesion at any concentration tested, indicating that the intrinsicheparin binding activity of Cyr61 is essential for mediating fibroblastadhesion.

Experiments designed to examine the effect of deleting thecarboxy-terminal domain of Cyr61 on Cyr61-mediated adhesion of 1064SKfibroblasts yielded consistent results in establishing that thecarboxy-terminal domain, containing the heparin binding site, werenecessary for fibroblast adhesion to Cyr61. The experiments followed theprotocol described above, but the recombinant human cyr61 constructencoded Cyr61 NT, a Cyr61 polypeptide containing amino acids 1-128 ofSEQ ID NO:4 (i.e., lacking the carboxy-terminal containing acid residues282-381 of SEQ ID NO:4). Thus, mature Cyr61 NT contains domains I, IIand III, corresponding to the IGFBP homology domain, the von Willebrandfactor type C repeat domain, and the thrombospondin type I repeatdomain, respectively, while lacking the heparan binding domain IV.Results of the experiments demonstrated that Cyr61 NT did not supportfibroblast adhesion, although this Cyr61 polypeptide fragment didsupport fibroblast migration, as described above (see Example 14).Further, immunological analyses showed that an antibody (i.e., GoH3)specifically recognizing the integrin α₆ blocked human fibroblastadhesion to Cyr61 polypeptides.

The following experiment showed that the requirement for Cyr61 heparinbinding sites was distinct from Cyr61-mediated adhesion through theα_(V)β₁ integrin. Washed HUVE cells were detached by 2.5 mM EDTA andresuspended in serum-free F-12K medium at 5×10³ cells/ml. Cells werepre-incubated with 2.5 mM EDTA, 1 mM RGD peptide, or 40 μg/ml anti-α₆β₃monoclonal antibody (LM609) at room temperature for 30 minutes, thenplated on wild-type Cyr61 (5 μg/ml), double-mutant Cyr61 (DM Cyr61 10μg/ml), or vitronectin (0.5 μg/ml). Immobilized cells were stained withmethylene blue and absorbances (A₆₂₀) were recorded. relative bindingcapacities (binding of cells not exposed to a pre-incubation compounddefined as 100%) are presented in Table III.

TABLE III Pre-incubation Cyr61 DM Cyr61 Vitronectin None 100 100 100EDTA 22 23 11 RGD peptide 50 23 13 Anti-α_(v)β₃ antibody 42 23 89The data were based on three independent trials. Thus, DM still mediatedHUVEC binding, establishing that the failure of DM to bind tofibroblasts was specific to that cell type.

Cell adhesion assays were also performed on smooth muscle cells. Bovineaortic smooth muscle cells (BASM cells) were subjected to the celladhesion assay described above in the context of assaying fibroblasts.Results showed that Cyr61 polypeptides induced adhesion of BASM cells.Heparin, but not “RGD” peptides (see, e.g., SEQ ID NO:31), inhibitedCyr61-induced adhesion of the smooth muscle cells. To identifyparticular integrin, receptors mediating the Cyr61 induction of BASMcell adhesion, antibody studies were conducted using anti-integrinantibodies recognizing specific integrins. Antibody GoH3, recognizingintegrin α₆, completely abolished BASM cell adhesion. Similarly,antibody JBla, specifically recognizing integrin β₁, eliminatedCyr61-induced BASM cell adhesion. Thus, integrin α₆β₁ mediatesCyr61-induced adhesion of smooth muscle cells. It is expected that Cyr61polypeptides that include the heparan binding domain IV will induceadhesion of any mammalian smooth muscle cell.

Accordingly, another aspect of the invention is directed to a method ofscreening for a modulator of cell adhesion comprising the steps of: (a)contacting a first fibroblast cell with a suspected modulator of celladhesion and a biologically effective amount of an ECM signalingmolecule-related biomaterial selected from the group consisting of aCyr61, a Fisp12, a CTGF, a NOV, an ELM-1 (WISP-1), a WISP-3, a COP-1(WISP-2), and fragments, analogs, and derivatives of any of theaforementioned members of the CCN family of proteins; (b) separatelycontacting a second fibroblast cell with a biologically effective amountof an ECM signaling molecule-related biomaterial described above,thereby providing a control; (c) measuring the level of cell adhesionresulting from step (a) and from step (b); and (d) comparing the levelsof cell adhesion measured in step (c), whereby a modulator of celladhesion is identified by its ability to alter the level of celladhesion when compared to the control of step (b). Preferably, thefibroblast cells present the α₆β₁ integrin. Also preferred arefibroblast cells that present a sulfated proteoglycan, such as heparansulfate proteoglycan. Any one of a number of CCN polypeptides may beused in the methods of the invention, such as Cyr61 (mouse-SEQ ID NO:2,human-SEQ ID NO:4, rat-Genbank Acc. No. AB015877), Fisp12/CTGF(mouse-SEQ ID NO:6, human-SEQ ID NO:8, N. viridescens-Genbank Acc. No.AJ271167, Sus scrofa-Genbank Acc. No. U70060, X. laevis-Genbank Acc. No.U43524, B. taurus Genbank Acc No. AF000137, and rat-Genbank Acc. No.AF120275), NOV (human-Genbank Acc. No. NM_(—)002514, mouse-Genbank Acc.No. Y09257, and G. gallus-Genbank Acc. No. X59284), ELM-1 (Wisp-1;human-Genbank Acc. No. NM_(—)003882, mouse-Genbank Acc. No. AB004873),COP-1 (Wisp-2; human-Genbank Acc. No. NM_(—)003881, mouse-Genbank Acc.No. AF100778), and Wisp-3 (human-Genbank Acc. No. NM_(—)003880).

Further, the invention comprehends screens for modulators ofinteractions between ECM signaling molecules, such as human Cyr61polypeptides, and α₆β₁, a specific integrin receptor whose activitiesinclude, but are not limited to, mediating the adhesion of fibroblasts,smooth muscle cells and endothelial cells. The screening methods involvecontacting a Cyr61 polypeptide with α₆β₁, preferably found in acomposition including a mammalian cell membrane such as an endothelial,fibroblast or smooth muscle cell membrane, in the presence and absenceof a potential or suspected medulator of the Cyr61-integrin interaction.Detection of relative levels of interaction, preferably in the form ofdetecting elative levels of cell membrane (or whole cell) adhesion,leads to the identification of modulators. The invention further extendsto methods of treating conditions or disorders, such as diseases,associated with excessive or inadequate cell adhesion, such as variousforms of fibrosis, defective angiogenesis, tumor growth, tumormetastasis, granulation tissue disorders, inflammatory responses, andcertain muscle disorders known in the art. Treatment involves deliveryof a therapeutically effective amount of a Cyr61 polypeptide or amodulator of the Cyr61-α₆β₁ integrin receptor interaction to a mammalsuch as a human by any means known in the art.

The invention also contemplates analogous methods of screening formodula0tors of fibroblast cell migration or proliferation. The methoddescribed below identifies modulators of cell migration; the describedmethod applies to methods of screening for modulators of cellproliferation by substituting the parenthetically noted terms. A methodof screening for modulators of fibroblast cell migration comprises thesteps of: (a) contacting a first fibroblast cell with a suspectedmodulator of cell migration (proliferation) and a biologically effectiveamount of an ECM signaling molecule-related biomaterial selected fromthe group consisting of a Cyr61, a Fisp12, a CTGF, a NOV, an ELM-1(WISP-1), a WISP-3, a COP-1 (WISP-2), and fragments, analogs, andderivatives of any of the aforementioned members of the CCN family ofproteins; (b) separately contacting a second fibroblast cell with abiologically effective amount of an ECM signaling molecule-relatedbiomaterial described above, thereby providing a control; (c) measuringthe level of cell migration (proliferation) resulting from step (a) andfrom step (b); and (d) comparing the levels of cell migration(proliferation) measured in step (c), whereby a modulator of cellmigration (proliferation) is identified by its ability to alter thelevel of cell migration (proliferation) when compared to the control ofstep (b). Preferred embodiments of the methods of screening formodulators of either cell migration or cell proliferation involve theuse of fibroblasts presenting an α₆β₁ integrin and/or a sulfatedproteoglycan.

EXAMPLE 30 Cyr61-Mediated Regulation of Gene Expression

Soluble Cyr61 protein added to primary human foreskin fibroblasts inculture elicits significant changes in gene expression within 24 hours.In contrast, immobilized Cyr61 is significantly less efficient ininducing such expression changes. Among the changes that occur is: (1)upregulation of matrix degrading enzymes, including MMP1, MMP3, and uPA;and (2) downregulation of matrix protein genes such as the type Icollagen chain gene. Cyr61 also induces expression of theinflammation-related proteins Il-1 and IL-6. In addition, Cyr61 inducesa substantial upregulation of the VEGF1 (vascular endothelial growthfactor 1 or VEGF-A), a potent angiogenic factor, and VEGF3 (VEGF-C), animportant angiogenic factor for the lymphatic system. These geneexpression alterations indicate that Cyr61 is useful in screening assaysdesigned to identify modulators of angiogenesis through detection of aneffect on Cyr61 activity.

Purified, soluble, recombinant Cyr61 protein was added to cultures ofprimary human foreskin fibroblasts for 24 hours. From these cells, mRNAwas isolated for preparation of probes to hybridize to a multi-gene blot(Clontech Atlas Human Array 1.2 cat no. 7850-1) containing about 650genes. The levels of expression of these genes in Cyr61-treated cellswere compared to that of control cells. From this comparison, it wasfound that the expression of several groups of genes was altered:upregulation of matrix degrading enzymes including MMP1, MMP3, and uPA;downregulation of matrix protein genes such as the type I collagen chaingene; induction of the inflammation-related cytokines IL-1 and IL-6, andinduction of the angiogenic molecules VEGF1 and VEGF3. These findingshave been confirmed by Northern blot analysis showing increases ordecreases of the mRNAs in question in Cyr61-treated cells. A time courseof expression changes has been established. In particular, by 6 hoursafter its addition, Cyr61 has induced expression of VEGF-A mRNA and theinduction is still evident at 12 and 24 hours post-addition. Also at 12and 24 hours post-addition, Cyr61 has induced expression of VEGF-Apolypeptide. With respect to VEGF-C, induction of mRNA is clearlyevident 12 hours after addition of Cyr61 and the induction persists atleast through 24 hours post-addition. Cyr61 is expected to induce theexpression of VEGF-C polypeptide with similar kinetics.

Cyr61 appears to be the key mediator for the action of TGF-beta, whichinduces Cyr61 strongly and is known to regulate matrix proteinsynthesis. Using mouse embryo fibroblasts derived from Cyr61 knockoutmice (see Example 31), it was shown that Cyr61 mediated TGF-betafunction. Whereas TGF-beta can induce collagen expression in embryofibroblasts derived from a cyr61^(±) mouse (littermate of a cyr61^(±)mouse), it cannot do so in cells that have an insertional inactivationof cyr61 i.e., cyr61 knock-out cells). Also, TGF-beta can induce BEFGexpression in cyr61^(±) fibroblasts, but not in cyr61^(±) fibroblasts.

Stimulation of fibroblasts by serum is known to induce the expression ofmany genes. Whereas cyr61^(±) cells respond to scrum stimulation withthe induction of VEGF, cyr61^(±) cells do not exhibit serum induction ofVEGF (although the background expression level is the same). Thisfinding indicates that Cyr61 is the mediator of VEGF induction understimulation by serum growth factors, and confirms the ability of Cyr61to regulate VEGF expression.

To prove that Cyr61-mediated serum induction of VEGF occurs at thetranscriptional level, collagen I and collagen II promoters linked toreporter genes were transfected into cyr61 knock-out cells and controlcells. Consistent with the aforementioned results, transcription fromthe transfected gene constructs was induced by TGF-beta in control cellsbut not in knock-out cells.

Thus, when fibroblasts are stimulated, Cyr61 expression can lead to geneexpression changes that produce proteins for matrix degradation andremodeling, cytokines that are chemotatic for macrophages andlymphocytes, and growth factors for angiogenesis.

As noted above, Cyr61 polypeptides interact with the α₆β₁ integrinreceptor of fibroblasts. Additional gene expression studies have shownthat both Cyr61 NT (lacking the heparan binding domain IV) and dmCyr61(containing substitutions in the two consensus XBBXB heparan bindingmotifs at amino acids 280-290 (H1) and 305-310 (H2) of SEQ ID NO:4) donot induce the expression of the above-referenced genes (e.g., VEGF,MMP1, and MMP3), in contrast to the effects of wild-type Cyr61. Based onthese results, the heparan binding domain of Cyr61 appears to berequired for induction of gene expression in fibroblasts. It is expectedthat Cyr61 peptides such as TSP1 will inhibit gene expression resultingfrom Cyr61 induction mediated by the α₆β₁ integrin receptor. It is alsoexpected that Cyr61 will induce the expression of these genes, andrelated genes, in other mammalian cell types, and that the heparanbinding domain will be required for such induction regardless of celltype.

The identification of target genes regulated by Cyr61, including genesinvolved in matrix remodeling (wound healing, metastasis, etc),inflammation, and angiogenesis, privides indications of suitable targetsof therapy using Cyr61.

Thus, in accordance with these findings, another aspect of the inventionis a method of screening for a modulator of angiogenesis comprising thesteps of: (a) contacting a first endothelial cell comprising a cyr61allele with a suspected modulator of angiogenesis; (b) measuring theCyr61 activity of the first endothelial cell; (c) measuring the Cyr61activity of a second endothelial cell comprising a cyr61 allele; and (d)comparing the levels of Cyr61 activity measured in steps (b) and (c),thereby identifying a modulator of angiogenesis.

A related aspect of the invention is drawn to a method of screening fora modulator of angiogenesis comprising the steps of: (a) contacting afirst endothelial cell with a polypeptide selected from the groupconsisting of a Cyr61, a Fisp12, a CTGF, a NOV, an ELM-1 (WISP-1), aWISP-3, a COP-1 (WISP-2), and fragments, analogs, and derivatives of anyof the aforementioned members of the CCN family of proteins; (b) furthercontacting the first endothelial cell with a suspected modulator ofangiogenesis; (c) contacting a second endothelial cell with thepolypeptide of step (a); (d) measuring the angiogenesis of the firstendothelial cell; (e) measuring the angiogenesis of the secondendothelial cell; and (f) comparing the levels of angiogenesis measuredin steps (d) and (c), thereby identifying a modulator of angiogenesis.

Another aspect of the invention is drawn to methods of treatingconditions or disorders, such as diseases, associated with gene under-or over-expression by delivering a biologically or therapeuticallyeffective amount of an ECM Signaling Molecule (e.g., a Cyr61polypeptide, Fisp12, CTGF), or modulator of a Cyr61-integrin receptorinteraction, using delivery means known in the art. A therapeuticallyeffective amount of an ECM Signaling Molecule is that amount thatresults in mitigation of the gene under- or over-expression. Genes whoseexpression can be affected by an ECM Signaling Molecule include, but arenot limited to, MMP1, MMP3, uPA, the type I collagen chain gene, theinflammation-related cytokines IL-1 and IL-6 and the genes encoding theangiogenic molecules VEGF1 and VEGEF3. Exemplary conditions, disordersand diseases include excessive or inadequate cell adhesion, such asvarious forms of fibrosis, defective angiogenesis, tumor growth, tumormetastasis, granulation tissue disorders, inflammatory responses, andcertain muscle disorders known in the art.

The methods of identifying modulators of angiogenesis take advantage ofthe potential for modulators to influence angiogenesis by affecting theactivity levels of a CCN protein such as Cyr61 by either influencing thelevel of expression of the protein or by influencing the specificactivity of the expressed protein.

EXAMPLE 31 Cyr61 Knock-out Mice

The mouse cyr61 gene was insertionally inactivated (i.e., knocked out)in vivo by targeted gene disruption and the phenotypes of heterozygousand homozygous knock-out mice were examined. Heterozygous mice(cyr61^(±)) appeared to be normal, as these mice did not exhibit anyapparent phenotype. The cyr61^(±) homozygous mice, however, exhibitedsevere vascular defects and apparent neuronal defects as well. Most ofthe cyr61^(±) mice died in utero, starting from E10.5 throughparturition, with most embryos dying around E13.5. There is a spectrumof developmental defects and phenotypes at the time of embryonic death.

The initial step in preparing knock-out mice was to construct atargeting vector that contained the mouse cyr61 gene insertionallyinactivated by introducing the bacterial lacZ gene encodingβ-galactosidase, which facilitated screening for knock-out mice. Acommercially available 129 Sv1 mouse genomic DNA library (Stratagene)was screened with a cyr61 probe and Clone 61-9 was identified. Clone61-9 phage DNA was then prepared and digested with Stul and BamHI usingconventional techniques. The 6 kb fragment containing the cyr61 promoterand coding region was ligated to a blunt-ended Kpnl linker, therebyattaching the linker to the Stul site. The fragment was then digestedwith BamHI and Kpnl and inserted into BamHI, Kpnl digested pBluescriptKS+. The recombinant pBluescript KS+ was cut with Smol and then ligatedto an Xhol linker. After linker ligation, the recombinant plasmid wascut with Xhol and the Xhol fragment bearing the lacZ coding region frompSAβgal (Friedrich et al., Genes Dev. 5:1513-1532 (1991)) was inserted.The PGK-TK-blue plasmid containing a thymidine kinase gene driven ty thePGK promoter (Mansour et al., Nature 336:348-352 (1988)) was cut withEcoRI and the ends were blunted with Klenow. The blunt-ended fragmentwas then ligated to Kpnl linkers. Finally, the cyr61-βgal-neo DNA andthe modified PGK-TK DNA were each cut with Kpnl and ligated to generatep61geo, the final targeting construct. Thus, p61geo contained functionalβ gal and neo coding regions flanked on the 5′ side by a 1.7 kb fragmentcontaining an intact cyr61 promoter and flanked on the 3′ side by a 3.7kb fragment containing the 3′ end of the cyr61 coding region (exons 2-5and 3′ flanking sequence). Homologous recombination of this insert intothe mouse chromosome would disrupt the cyr61 coding region and place theβgal and neo coding regions into the genome.

Cell culturing was performed according to Genome Systems instructionsfor mouse embryonic fibroblasts (MEFs), or as described by Li et al.,Cell 69:915-926 (1992), with modifications, for Jl ES cells. Briefly,MEFs were cultured in 7.5% CO₂ in an incubator at 37° C. with DMEM (highglucose) medium (Gibco/BRL#11965-084) and 10% heat-inactivated FetalCalf Serum (HyClone), 2 mM glutamine 0.1 mM non-essential amino acids,and optionally with 100 U of Penicillin/Streptomycin. MEFs were isolatedfrom mouse embryos at E14.5 and supplied at passage 2.

For feeder cells, MEFs were mitotically inactivated by exposure to 10μg/ml Mytomycin C(Sigma) in culture medium at 37° C. (7.5% CO₂) for 2-5hours. Cells were then washed 3 times with PBS. Mitotically inactivatedMEFs were harvested with trypsin-EDTA(Gibco/BRL) and plated about1×10³/cm² with MEF medium.

Jl embryonic stem(ES) cells were cultured in DMEM (no pyruvate, highglucose formulation; Gibco/BRL# 11965-084) supplemented with 15% heatinactivated FCS (Hyclone), 2 mM glutamine (Gibco/BRL), 0.1 mMnon-essential amino acids(Gibco/BRL), 10 mM HEPES buffer (Gibco/BRL), 55μM β-mercaptoethanol (Gibco/BRL), and 1,000 U/ml ESGRO (leukemiainhibitory factor, LIF)(Gibco/BRL). Jl cells were routinely cultured inES medium on a feeder layer of mitotically inactivated MEFs in ahumidity saturated incubator at 37° C. in 7.5% CO₃. Normally, 1.5×10⁶ Jlcells were seeded in a 25 cm² tissue culture flask and the medium waschanged every day. Cell cultures were divided 2 days after seeding,usually when the flask was about 80% confluent. To dissociate ES cells,cells were washed twice with PBS (Ca- and Mg-free) and trypsinized withTrypsin/EDTA at 37° C. for 4 minutes. Cells were than detached, mixedwith trypsin/EDTA thoroughly, and incubated for an additional 4 minutes.The cell suspension was then pipetted several (20-30) times to break upthe cell clumps. A complete dissociation of cells was checkedmicroscopically. ES cells were frozen with ES medium having 10% FCS and10% DMSO(Sigma) at about 4-5×10⁶ cells/ml, with 0.5 ml/tube. Frozencells were stored at −70° C. overnight and transferred into liquidnitrogen the next day. Frozen cells were quickly thawed in a 37° C.water bath, pelleted in 5 ml ES medium to remove DMSO, and plated in 25cm² flasks with fresh MEF feeder cells.

To transfect mouse cells with a transgene, the p61 geo targetingconstruct was linearized by Not1 digestion, suspended in PBS at 1 μg/ml,and introduced into Jl ES cells by electroporation. Rapidly growing(subconfluent, medium newly refreshed) Jl ES cells were trypsinized,counted, washed and resuspended in the electroporation buffer containing20 mM HEPES, pH 7.0, 137 mM NaCl, 5 mM KCl, 6 mM D-glucose, and 0.7 mMNa₂HPO₁, at 1×10⁷ cells/ml. Linearized DNA was added to the cellsuspension at 45 μg/ml, mixed, and incubated at room temperature for 5minutes. A 0.8 ml aliquot of cell-DNA mix was then transferred to acuvette and subjected to electroporation with a BioRad Gene Pulser usinga single pulse at 800 V, 3 μF. Cells were left in the buffer for 10minutes at room temperature, and then plated at 4×10⁶ cells/100 mm platewith neomycin-resistant MEF feeder cells. Cells were then cultured understandard conditions without drug selection. After 24 hours, selectionmedium containing ES medium supplemented with 400 μg/ml (total) G418(Gibco/BRL) and 2 μM Ganciclovir (Roche) was substituted. Selectionmedium was refreshed daily. Individual colonies were placed inmicrotiter wells and cells were dissociated with 25 μl 0.25%trypsin-EDTA/well on ice and subsequently incubated in a humidifiedincubator at 37° C. with 7.5 CO₂, for 10 minutes. Cell suspensions werethen mixed with 25 μl ES medium and pipetted up and down 10 times tobreak up clumps of cells. The entire contents of each well were then wastransferred to a well in a 96-well flat-bottom dish with 150 μl of ESmedium in each well and grown using conventional culturing techniquesfor 2 days.

Confluent ES cell clones were washed and treated with lysis buffer (10mM Tris (pH 7.7), 10 mM NaCl, 0.5% (w/v) sarcosyl, and 1 mg/mlproteinase K) in a humid atmosphere at 60° C. overnight. After lysis, amixture of NaCl and ethanol (150 μl of 5 M NaCl in 10 ml of coldabsolute ethanol) was added (100 μl/well) and genomic DNA was isolated.The genomic DNA of each ES cell clone was digested with EcoRI (30μg/well) and subjected to Southern blot assay.

Southern blotting was preformed as described in “Current Protocols inMolecular Biology” (Ausubel et al., [1999]). Briefly, EcoRI fragments ofgenomic DNA were fractionated by electrophoresis through 0.8% agarosegels and blotted onto nylon membranes (Bio-Rad) by downward capillarytransfer with alkaline buffer (0.4 M NaOH). The probes, a BumHI-EcoRIfragment 3′ to the long arm of the targeting construct (p61geo) or theneo coding region sequences, were prepared by random primer labeling(Prim-it II, Stratagene) using [α-³²P] dCTP (NEN). Membranes wereprehybridized in hybridization buffer (7% SDS, 0.5 M NaHPO₄ (pH 7.0),and 1 mM EDTA) at 65° C. for 15 minutes in a rolling bottle. Freshhybridization buffer was added with the probe and membranes werehybridized for 18 hours. Hybridized membranes were briefly rinsed in 5%SDS, 40 mM NaHPO₄ (pH 7.0), 1 mM EDTA and then washed for 45 minutes at65° C. with fresh solution. This wash solution was replaced with 1% SDS,40 mM NaHPO₄ (pH 7.0), 1 mM EDTA and washed twice for 45 minutes at 65°C. with fresh solution. After washing, membranes were exposed to ascreen, which was then scanned using a Phosphorimager (MolecularDynamics). Blots were routinely stripped and re-probed with the controlneo probe to ensure that random integration had not occurred, usingconventional techniques.

Results of the Southern analysis showed that the genomic DNA of 14colonies (231 colonies examined) contained a mutant cyr61 allele in alocation consistent with integration via homologous recombination. Thesizes of the detected fragments were 6.4 kb for the wild-type cyr61allele and 7.4 kb for the mutant allele with the cyr61 probe, no brandfor the wild-type cyr61 allele and a 7.4 kb band for the mutant allelewith the neo probe.

Genotyping was also done by PCR using a RoboCycler (Stratagene). Primerswere designed to amplify a 2.1 kb DNA fragment from mutant alleles. ThePCR product covers the 5′-flank of the short arm of the targetingconstruct through to the sequence of lazZ (β-gal) within the targetingconstruct. The upper PCR primer acquence was 5′-CACAACAGAAGCCAGGAACC-3′(SEQ ID NO:24) and the lower PCR primer sequence was5′-GAGGGGACGACGACAGTATC-3′ (SEQ ID NO:25). PCR reaction conditions were95° C. for 40 seconds, 63° C. for 40 seconds, and 68° C. for one minute,for 35 cycles.

For genotyping mouse tails or embryo tissues, two sets of primers wereincluded in the same PCR reaction to amplify both wild-type and mutantalleles. A single upper PCR primer (b) was used, which had the sequence5′-CAACGGAGCCAGGGGAGGTG-3′ (SEQ ID NO:26). The lower PCR primer foramplifying the wild-type allele, lower wt primer, had the sequence5′-CGGCGACACAGAACCAACAA-3′ (SEQ ID NO:27) and would amplify a fragmentof 388 bp. The lower PCR primer for amplifying the mutant allele was thelower mutant primer and had the sequence 5′-GAGGGGACGACGACAGTATC-3′ (SEQID NO:28); a 600 bp fragment was amplified from mutant alleles. Reactionconditions were 95° C. for one minute, 63° C. for one minute, and 72° C.for one minute, for 30 cycles.

PCR amplification of mutant alleles of cyr61 using the mutant-specificprimers produced a fragment of 2.1 kb and attempts to amplify thewild-type allele with those primers failed to produce a detectablyamplified fragment, in agreement with expectations. Southern analysesidentified a 7.4 kb band (mutant allele) and a 6.4 kb band (wild type)in heterozygous mutants; only the 6.4 kb band was detected when probingwild-type DNAs. Both the PCR data and the Southern data indicate thatmutant cyr61 alleles were introduced into the mouse genome in a mannerconsistent with homologous recombination.

The selected ES cell clones were then expanded for micro-injection intoE3.5 blastocysts from C57BL/6J mice. Embryo manipulations were carriedout as described by Koblizek et al., Curr. Biol. 8:529-532 (1998) andSuri et al., Science 282;468-471 (1998), with modifications. Briefly,the Jl ES cell clones were harvested and dissociated with trypsin-EDTA.The cells were resuspended in CO₂-independent medium (Gibco-BRL) with10% FBS and kept on ice. About 15-20 ES cells were injected into eachblastocyst from C57BL/6J (Jackson Labs), injected blastocysts werecultured for 1-2 hours prior to transfer into the uterine horns ofpseudopregnant foster mothers (CD-1, Harlan). Chimeras were identifiedby coat color. Male chimeras with a high percentage of agouti coat colorwere caged with C57BL/6J females to test germ-line transmission of theES-cell genotype F, offspring carrying the targeted (i.e., mutant)allele were then back-crossed with C57BL/6J females for a few rounds toestablish an inbred C57BL genetic background. In addition, a mutantmouse line having the inbred 129SvJ genetic background was obtained bymating germ-line chimera males with 129SvJ females.

Five ES cell clones were injected and generated chimeric offspring withES cell contributions ranging from 30%-100%, as judged by the proportionof agouti coat color. Four and two chimeric males derived from ES cellclones 4B7 and 2A11, respectively, efficiently transmit the targetedallele through the germline. The cyr61 heterozygous mutant mice appearedhealthy and fertile. The 4B7 chimeric line was either bred to 129SvJmice to maintain the targeted allele in a SvJ129 genetic background, orback-crossed with C57BL/61 mice to transfer the mutation into theC57BL/61 background. The 2A11 targeted line was maintained in the 129SvJgenetic background. Similar phenotypes were exhibited by the 4B7₁₂₉,4B7_(CSTBL), and 2A11 mouse lines.

Among the offspring from intercrosses of cyr61^(±) mice that wereexamined, 141 were cyr61^(±), 225 were cyr61^(±), and no homozygouscyr61^(±) mice were observed at this age, except that 10 cyr61^(±) pupswere born alive and died within 24 hours of birth. Based on Mendelianratios, the majority (>90%) of the cyr61^(±) animals should have diedbefore birth. Thus, staged prenatal fetuses were examined by PCR, addescribed above. Starting from E10.5, the numbers of homozygous mutantembryos were found to be less than expected based on a Mendelian ration,which might have been due to resorption of homozygous mutant embryos.However, most (80%) of the E10.5 cyr61^(±) embryos appeared normalcompared to littermates. At this stage (E10.5), the failure ofchorioallantoic fusion was found in some embryos and this phenotyperesulted in early embryonic lethality. The allantois of this type ofembryo appeared ball-shaped and often was filled with blood. While noother defects were specifically identified, hemorrhage began to appearin a few of the cyr61-null embryos.

At E11.5, about 50% of cyr61^(±) embryos were indistinguishable fromwild-type or heterozygous mutant littermates by appearance. By E11.5,embryos lacking a chorioallantoic fusion were consistently deteriorated.Increasing numbers and severity of hemorrhage were also observed incyr61-null embryos. Hemorrhages occurred in different areas, includingthe placenta, intra-uterus, intra-amnion, embryo body trunks, bodysides, and head. At this stage, placental defects were also found insome null mutant embryos. The placentae associated with these embryosshowed a sub-standard vasulature network. Unlike the early lethalityassociated with the failure of chorioallantoic fusion, embryos withplacental defects typically lived and developed normally.

At E12.5, cyr61^(±) embryos still presented three phenotypes: 1)unaffected, 2) alive with hemorrhage and/or placental defects, and 3)deteriorated, though with the proportion of categories changed fromearlier stages. About 30% of the cyr61-null embryos remained unaffectedat this stage. About 50% of the null mutant embryos showed signs ofhemorrhage and/or placental defects and 20% of this type of embryo didnot survive the vascular or the placental defects. About 20% ofcyr61^(±) embryos did not have a chorioallantoic fusion and died at muchearlier stages, as judged by the under-development of defective embryos.

By E13.5, none of the cyr61^(±) embryos that had shown hemorrhage,placental defects, or failure of chorioallantoic fusion were alive,although about 30% of the total Cyr61-deficient embryos showed noapparent phenotype. Embryos examined at later stages (>E14.5) showed thesame phenotypic pattern and the same proportion for each type of defect,but with increasing severity.

Additional investigation, at the cellular and sub-cellular levels, wasperformed using the following techniques. MEF cells were harvested asdescribed by Hogan et. al., Manipulating the Mouse Embryo. A LaboratoryManual (1994). Briefly, E11.5 embryos from crosses of two heterozygouscyr61-targeted parents were dissected in DMEM without serum. The limbs,internal organs, and brain were removed. Embryo carcasses were thenminced with a razor blade and dissociated with trypsin/EDTA at 37° C.with rotation for 10 minutes. Half of the dissociation buffer was thenadded to an equal volume of DMEM plus 10% FBS. Dissociation andcollection steps were repeated five times. Collected cells were expandedand split at a 1:10 ratio to select the proliferating fibroblast cells.

To prepare total cell lysates, a 100 mm plate of MEF cells was culturedto near confluency. Cells were activated with fresh medium containing10% serum and incubated at 37° C. for 1.5 hours before being harvested.Cells were then washed and centrifuged using conventional procedures.The cell pellets were resuspended in 100 μl RIPA buffer (0.5% sodiumdeoxycholate, 0.1% SDS, 1% Nonidet P-40, 50 mM Tris-Cl, pH 8.0, 150 mMNaCl, aprotinin 0.2 units/ml, and 1 mM PMSF) and put on ice for 10minutes to lyse the cells. The cell suspension was centrifuged and thesupernatant (cell lysate) was stored at −70° C. for further analysis.One third of the supernatant was subjected to Western blot analysisusing a TrpE-mCyr61 polyclonal anti-serum.

To confirm that homozygous cyr61^(±) animals did not express Cyr61, MEFcells were prepared from E11.5 embryos resulting from intercrosses oftwo cyr61^(±) parents. Cell lysates were collected from serum-stimulatedMEFs of different genotypes and were subjected to Western blot analysesusing anti-Cyr61 antiserum (trpE-mCyr61). The Western blot demonstratedthat the Cyr61 protein level was not detectable in KO (knockout) MEFcells, while heterozygous cyr61^(±) cells expressed the Cyr61 protein athigh levels under the same culture conditions and serum stimulation. Thelack of expression of Cyr61 in cyr61^(±) animals was further confirmedby Northern blot analyses, in which cyr61 mRNA was not detectable inserum-induced KO MEF cells. Thus, the null mutation of cyr61^(±) hasbeen confirmed as eliminating Cyr61 expression at both the mRNA andprotein levels.

Defects in placental development, a major cause of embryonic death incyr61^(±) mice, were further analyzed. Histological analyses of mouseplacentac generally followed Suri et al., (1998). Briefly, placentaefrom E12.5 embryos were dissected in cold PBS and fixed with 4%paraformaldehyde in 0.1 M phosphate buffer (PB) at 4° C. for overnight.Fixed placentae were then dehydrated through increasing concentrationsof alcohol (50%, 75%, 90%, 95%, and 100%) two times. Dehydrated tissuewas then cleared with Hemo-De (a xylene alternative), 1:1ethanol/Hemo-De (Fisher), and 100% Hemo-De, and the clearing process wasrepeated. Cleared tissues were then equilibrated in a 1:1 mixture ofparaffin:Hemo-De at 60° C. for one hour in a vacuum oven and the processwas repeated. Tissues were embedded in paraffin with Histoembedder(Leica). The paraffin-embedded placentae were cut into 10 μm slices witha nicrotome (Leica). Finally, tissue sections were subjected to Harris'Hematoxylin and Eosin staining (Asahara et al., Circ. Res. 83:233-240[1998]).

Placentae for immunohistochemical staining were dissected in cold PBSand fixed in 4% paraformaldehyde at 4° C. overnight. Fixed tissue wastransferred to 30% sucrose in PBS at 4° C. overnight. Placentae werethen embedded in OC.T. (polyvinyl alcohol, carbowax solution) on dryice. Frozen blocks were stored at −70° C. or cut into 7 μm sections witha cryotome (Leica). Immunohistochemical staining was done as recommendedby the manufacturer (Zymed). Briefly, frozen sections were post-fixedwith 100% acetone at 4° C. for 10 minutes. Endogenous peroxidase wasblocked with Peroxo-Block (Zymed). Sections were incubated with a 1:250dilution of biotinylated rat anti-mouse PECAM-1 (i.e., plateletendothelial cell adhesion molecule-1) monoclonal antibody MEC 13.3(Pharmingen) at 4° C. overnight. A Histomouse-SP kit with Horse RadishPeroxidase (Zymed) was used to detect PECAM-1 signals.

The results of histological and immunohistochemical analyses showed thatCyr61-null placentae contained a limited number of embryonic blood cellsand were largely occupied by maternal blood sinuses. Abnormally compacttrophoblastic regions were also observed. PECAM-1 staining demonstratedthe highly-vascularized labyrinthine zone in a heterozygous mutantplacenta. Under higher magnification, flows of fetal blood cells withinthe PECAM-1 stained vessels were identified. Consistent with thevariation in phenotypes among the Cyr61-deficient embryos, the stainingof placentae from numerous Cyr61^(±) embryos also reflected placentaldefects to various degrees. Nonethless, the placental defects observedwith PECAM-1 staining can be classified into two groups, groups I andII. Group I of type-II (type I-embryos with complete failure ofchorioallantoic fusion not surviving E10.5, type II-embryos withpartially defective chorioallantoic fusion surviving through aboutE13.5) exhibits a set of placental defects that is characterized by thevirtual absence of embryonic vessels, the presence of condensedtrophoblasts, and the presence of a compressed labyrinthine zone. Ahigher magnification view confirms that no vessels developed in thelabyrinthine with placental defects of this kind. Placentae with a groupII defect showed fair amounts of PECAM-1-positive staining and condensedcapillary structures. However, the PECAM-1-stained vessel-likestructures were degenerated and collapsed, with no fetal blood cellsinside.

Thus, the lack of Cyr61 causes two types of placental defects. In typeI, the failure of chorioallantoic fusion results in the loss of physicalconnection between the embryo and the placenta. In type II placentaldefects, the physical connection is established by successfulchorioallantoic fusion. However, the embryonic vessels only reach to thesurface of the placenta or, with successful penetration through thechorionic plate, develop an immature non-functional vascular structurein the labyrinthine zone.

X-gal staining was also used to assess embryonic development in variousxyr61 backgrounds. (The targeting DNA, p61 geo, was designed to knockout the Cyr61 gene and also to “knock in” in β-gal gene as a marker toreflect the expression of Cyr61). X-gal (i.e.,5-Bromo-4-chloro-3-indolyl-β-D-galactopyranoside) staining forβ-galactosidase expression was performed on heterozygous cyr61^(±)embryos staged from E9.5 to E11.5. The staining was done as described(Suri et al., [1998]). Staged embryos were fixed in a 0.2%paraformaldehyde solution at 4° C. overnight. Fixed tissue was incubatedin 30% sucrose in PBS plus 2 mM MgCl₂ at 4° C. overnight. Tissue wasthen embedded in OCT on dry ice and cut with a cryotome into 7 μmsections. Frozen tissue sections were post-fixed in 0.2%paraformaldehyde and stained with X-gal (1 mg/ml) at 37° C. for 3 hoursin the dark. Slides were counter-stained with 1% Orange G. Stainedslides were then serially dehydrated through increasing concentrationsof methanol, cleared with Hemo-De, and slides were mounted.

X-gal staining of the E9.5 embryos, including the extra-embryonictissues, showed β-galactosidase expression, driven by the cyr61promoter, at the tip of the allantois adjacent to the chorion in thechorioallantoic placenta. The staining of more advanced E10.5 embryosillustrated that large vessels branching from the allantoic vessels weredeveloped in the chorionic plate and could easily be identified in theendothelial lining using X-gal. Further developed E11.5 placenta showedthe same expression pattern as E10.5 embryos. While the staining washighly associated with the endothelium of the umbilical and chorionicvessels, no detectable staining in the labyrinthine zone, where amicrovasculature network was developing, was seen at E11.5. The presenceof Cyr61 in the allantois al, and proximal to, the fusion surface withthe chorion, and in the umbilical and chorionic vessels, furthersupports the important role of Cyr61 in angiogenesis. Cyr61 was involvedin chorioallantoic fusion and was critical for proper angiogenicdevelopment as placentation progressed. Moreover, a staining of theE11.5 embryo confirmed that Cyr61 was expressed in the paired dorsalaortae and the major arteries branching from the heart, which isconsistent with the hemorrhaging seen in Cyr61-null mutants.

Apparent from the preceding description is another aspect of theinvention, which is directed to a method of screening for modulators ofangiogenesis comprising the steps of: (a) constructing a non-humantransgenic animal comprising a mutant allele of a gene encoding apolypeptide selected from the group consisting of a Cyr61, a Fisp12, aCTGF, a NOV, and ELM-1 (WISP-1), a WISP-3, a COP-1 (WISP-2); (b)contacting the transgenic animal with a suspected modulator ofangiogenesis; (c) further contacting a wild-type animal of the samespecies with the polypeptide, thereby providing a control; (d) measuringthe levels of angiogenesis in the transgenic animal; (e) measuring thelevel of agiogenesis of the wild-type animal; and (f) comparing thelevels of angiogenesis measured in steps (d) and (e), therebyidentifying a modulator of angiogenesis.

Transgenic animals are characterized as described above and, based onsuch characteriztions, a variety of genotypes may be usefully employedin the methods of the invention. For example, the transgenic animal maybe either homozygous or heterozygous and the mutant allele may result inno expression (i.e., a null mutation) or altered activity levels. Apreferred transgenic animal is a mouse, although any non-humanvertebrate organism may be used, including other mammals (e.g., rat,rabbit, sheep, cow, pig, and horse, among others) or birds (e.g.,chicken). A preferred transgene is an insertional inactivation, orknock-out, of a gene encoding a CCN protein (e.g., cyr61); alsopreferred is an insertional inactivation resulting from theintroduction, or “knocking in”, of an identifiable marker gene such aslacZ encoding β-galactosidase. Of course, many transgene constructionsare possible, including transgenes resulting from the replacement ofwild-type sequence by related sequences that specify variant amino acidsequences. It should be understood from the preceding discussion thatthe invention comprehends gene therapy approaches involving theintroduction of a transgene into a cell to treat any of a variety ofconditions or disorders, such as diseases.

Also apparent from the preceding description is another aspect of theinvention, which is drawn to a mammalian cell comprising a cyr61mutation selected from the group consisting of an insertionalinactivation of a cyr61 allele and a deletion of a portion of a cyr61allele. The mammalian cell is preferably a human cell and the mutationis either heterozygous or homozygous. The mutation, resulting frominsertional inactivation or deletion, is either in the coding region ora flanking region essential for expression such as a 5′ promoter region.Cells are also found associated with non-human animals.

EXAMPLE 32 Adhesion to Platelets and Macrophages

Platelet immobilization plays an important role in wound healing, forexample by contributing to thrombosis in the process of stanching theflow of blood. Proteins of the CCN family, such as Cyr61 andFisp12/CTGF, promote platelet adhesion by interacting with the α_(llb)β₃integrin.

Recombinant Cyr61 and Fisp12/mCTGF, synthesized in a Baculovirusexpression system using Sf9 insect cells, were purified from serum-freeconditioned media by chromatography on Sepharose S as described (Kireevaet al., [1997]; Kireeva, et al., [1996]). SDS-PAGE analysis of purifiedCyr61 and Fisp12/mCTGF revealed the presence of single CoomassieBlue-stained bands of 40-kDa and 38-kDa, respectively. On immunoblots,the purified proteins reacted specifically with their cognateantibodies. Protein concentrations were determined using the BCA proteinassay (Pierce) with bovine serum albumin (BSA) as the standard.

Microtiter wells were coated with purified recombinant Fisp12/mCTGF orCyr61, and the adhesion of isolated platelets to these proteins wasdetected with ¹²⁵I-mAb15, an anti-β₃ monoclonal antibody (Frelinger etal., J. Biol. Chem 265:6346-6352 [1990]). Platelets were obtained fromvenous blood drawn from healthy donors and collected intoacid-citrate-dextrose (ACD). Washed platelets were prepared bydiffenential centrifugation as described (Kinlough-Rathbone et al.Thromb. Haemostas 2:291-308 [1997]), and finally resuspended inHEPES-Tyrode's buffer (5 mM HEPES, pH 7.35, 1 mM MgCl₂, 1 mM CaCl₂, 135mM NaCl, 2.7 mM KCl, 11.9 mM NaHCO₃, 1 mg/ml dextrose and 3.5 mg/mlBSA). The platelet concentration was adjusted to 3×10³ platelets/ml.

Microtiter wells (Immulon 2 removawell strips, Dynex Technologies, Inc.)were coated with Cyr61, Fisp12/mCTGF, or fibrinogen (25 μg/ml, 50μl/well) incubated overnight at 22° C., and then blocked with 3% BSA at37° C. for 2 hours. Washed platelets were added to the wells (100μl/well) in the presence and absence of platelet agonists and incubatedat 37° C. for 30 minutes. The wells were washed with HEPES-Tyrode'sbuffer and adherent platelets were detected with ¹²⁵I-mAb15, an anti-β₃monoclonal antibody. Exposure to the labeled antibody (50 mM, 50μl/well) proceeded for 1 hour at 22° C. After extensive washing withHEPES-Tyrode's buffer, bound radioactivity was determined by γ-counting.In inhibition studies, washed platelets were pre-incubated with blockingpeptides or antibodies at 37° C. for 15 minutes prior to addition tomicrotiter wells. In experiments to examine the effect of divalentcation chelation, EDTA (5 mM) was added to suspensions of washedplatelets and pre-incubated at 37° C. for 15 minutes.

The anti-β₃ antibody was radioiodinated with carrier-free Na¹²⁵I(Amersham Corp.) using the IODO-BEADS iodination reagent (Pierce) to aspecific activity of approximately 2 μCi/μg. This antibody binds equallyto α_(llb)β₃ present on activated (see below) and unactivated platelets.As controls, BSA- and fibrinogen-coated (KabiVitrum Inc.) wells werealso used. Initially, the adhesion of unactivated versus activatedplatelets to immobilized Fisp12/mCTGF and Cyr61 was compared. To ensurethat plateltes were not activated during the washing procedures, PGl₂(100 nM), which inhibits activation by raising platelet cAMP levels, wasadded to the platelet suspensions.

Unactivated platelets failed to adhere to either protein. However,activation of platelets with 0.1 U/ml thrombin, 500 mM U46619, or 10 μMADP caused a dramatic increase in platelte adhesion to bothFisp12/mCTGF- and Cyr61-coated wells. To confirm that the adhesionprocess was activation-dependent, PGl₂ (100 nM) was added with theagonists to prevent platelet activation. Under these conditions,platelet adhesion to both Fisp12/mCTGF and Cyr61 was significantlyinhibited.

For comparison, platelet adhesion to fibrinogen-coated wells wasassessed. While unactivated platelets were capable of adhering toimmobilized fibrinogen at a low level, platelet adhesion to Cyr61 andFisp12/mCTGF appeared to be absolutely dependent on cellular activation.Following platelet activation with strong agonists such as thrombin andU46619, platelet adhesion to Cyr61 and Fisp12/mCTGF was comparable tofibrinogen. The weaker agonist, ADP, caused a lesser response. BecauseADP does not induce secretion of α-granule proteins from washed humanplatelets and does not induce platelet aggregation in the absence ofexogenous fibrinogen, ADP was used to induce platelet adhesion insubsequent experiments.

To further substantiate the activation-dependent adhesion of plateletsto these proteins, an acid phosphatase assay designed to quantitate therelative numbers of adherent platelets was performed. This assaymeasured the acid phosphatase activity of adherent platelets. Followingthe adhesion and washing procedures described above, the substratesolution (0.1 mM sodium acetate, pH 5.0, 20 mM p-nitrophenylphosphate,and 0.1% Triton X-100; 150 μl/well) was added and incubated for 2 hoursat 37° C. The reaction was stopped by the addition of 20 μl 2N NaOH, andabsorbance at 405 nm was measured. Both the ¹²⁵I-mAb15 binding assay andthe acid phosphatase assay for adhesion of ADP-stimulated platelets tofibrinogen, Fisp12/mCTGF, and Cyr61, produced similar results. Becausethe amounts of bound ¹²⁵-mAb15 were directly proportional to thequantity of integrin α_(llb)β₃ on the adherent platelets, the acidphosphatase assay was used in subsequent studies.

The adhesion of ADP-activated platelets to Fisp12/mCTGF and Cyr61 wasdose-dependent and saturable. In the presence of PGl₂, unactivatedplatelets adhered poorly to both proteins, even at high coatingconcentrations. The specificity of the adhesion process wascharacterized in inhibition studies using anti-peptide polyclonalantibodies raised against the central variable regions of Fisp12/mCTGFand Cyr61. On immunoblots, rabbit polyclonal anti-Fisp12/mCTGF andanti-Cyr61, prepared as described in Example 29, reacted specificallywith Fisp12/mCTGF and Cyr61, respectively. No cross-reactivity wasobserved. In addition, anti-Fisp12/mCTGF antibody inhibited plateletadhesion to Fisp12/mCTGF, but not to Cyr61, and anti-Cyr61 antibodyinhibited Cyr61-mediated platelet adhesion but not that mediated byFisp12/mCTGF. No inhibition was observed with normal rabbit IgG. Also,neither anti-Fisp12/mCTGF antibody nor anti-Cyr61 antibody inhibitedplatelet adhesion to fibrinogen-coated wells. Thus, the abilities ofFisp12/mCTGF and Cyr61 to mediate platelet adhesion are intrinsicproperties of these proteins.

Upon platelet activation, the ligand binding affinities of integrinsα_(llb)β₃ and α_(V)β₃ are upregulated (Shattil, et al., Blood91:2645-2657 [1998]; Bennett, et al., J. Biol Chem. 272:8137-8140[1997]). To determine whether these integrin receptors mediated plateletadhesion to Fisp12/mCTGF and Cyr61, the inhibitory potentials of peptideantagonists and the divalent cation chelator, EDTA, were tested.Preincubation of platelets with EDTA at 37° C. completely abolishedplatelet adhesion to both proteins, indicating that the adhesion processwas divalent cation dependent. Cation dependency of adhesion isconsistent with the involvement of an integrin receptor.

The major platelet integrin, α_(llb)β₃, is sensitive to inhibition byRGD-containing peptides and the dodecapeptide H₁₂(H₂N—HHLGGAKQAGDV—CO₂H, SEQ ID NO:29, Research Genetics Inc.) derivedfrom the fibrinogen γ chain (Plow et al., Proc. Natl. Acad. Sci. (USA)82:8057-8061 [1985]; Lam, et al., J. Biol Chem. 262:947-950 [1987]). Theadhesion of ADP-activated platelets to Cyr61 and Fisp12/mCTGF wasspecifically inhibited by GRGDSP (SEQ ID NO:31), but not by GRGESP (SEQID NO:32). (Peninsula Laboratories). Likewise, the RGD-containing snakevenom peptide echistatin (Can et al., J. Biol Chem. 263:19827-19832[1988], Sigma Chemical Co.) also completely blocked platelet adhesion toboth proteins. It has also been shown that the dodecapeptide H₁₂preferentially interacts with integrin α_(llb)β₃ as compared to integrinα_(V)β₃ (Cheresh et al., Cell 58:945-953 [1989]; Lam et al., J. Biol.Chem. 264:3742-3749 [1989]). Thus, the finding that H₁₂ inhibitedplatelet adhesion to Cyr61 and Fisp12/mCTGF indicated that this processwas mediated by α_(llb)β₃ rather than α_(V)β₃. While thecomplex-specific monoclonal antibody AP-2 (anti-α_(llb)β₃, Pidard etal., J. Biol. Chem. 258:12582-12587 [1983]) completely blocked theadhesion of ADP-activated platelets to Fisp12/mCTGF and Cyr61, noinhibition was observed with LM609 (anti-α_(V)β₃; Cheresh et al., J.Biol. Chem. 262:17703-17711 [1987]) or with normal mouse IgG. In controlsamples, the adhesion of ADP-activated platelets to fibrinogen was alsocompletely inhibited by EDTA, RGDS, echistatin, H₁₂ or AP-2, but not byRGES or LM609. These results indicate that platelet adhesion to theseproteins is mediated through interaction with activated integrinα_(llb)β₃.

A solid-phase binding assay to detect the receptor-ligand interactionsshowed that α_(llb)β₃ binds directly to Fisp12/mCTGF and Cyr61. In theseexperiments, activated and unactivated α_(llb)β₃ were purified fromplatelet lysates. Activated α_(llb)β₃ was purified by RGD affinitychromatography, as described (Knezevic et al., J. Biol. Chem.271:16416-16421 [1996]). Briefly, outdated human platelets were isolatedby differential centrifugation and solubilized in lysis buffer (10 mMHEPES, pH 7.4, 0.15 M NaCl, containing 1 mM CaCl₂, 1 mM MgCl₂ 100 μMleupeptin, 1 mM phenylmethylsulfonyl fluoride, 10 mM-ethylmaleimide, and50 mM octylglucoside). The octylglucoside extract was incubated with 1ml GRGDSPK-coupled Sepharose 4B overnight at 4° C. After washing with 15ml column buffer (same as lysis buffer except it contained 25 mMoctylglucoside), bound α_(llb)β₃ was eluted with 1.7 mM H₁₂ (2 ml) incolumn buffer. The H₁₂ eluate was applied to a Sephacryl S-300 HighResolution column (1.5×95 cm), and α_(llb)β₃ was eluted with 10 mMHEPES, pH 7.4, 0.15 M NaCl, 1 mM CaCl₂, 1 mM MgCl₂ and 25 mMoctylglucoside.

Unactivated α_(llb)β₃ was isolated by the method of Fitzgerald et al.,Anal. Biochem. 151:169-177 (1985), with slight modifications. Theflow-through fraction of the GRGDSPK-Sepharose column was applied to aconcanavalin A-Sepharose 4B column (1×20 cm). Unbound proteins werewashed with 50 ml column buffer, and bound α_(llb)β₃ was then elutedwith 100 mM mannose dissolved in column buffer. Fractions containingα_(llb)β₃ were further purified on a Sephacryl S-300 High Resolutioncolumn

To perform the solid-phase binding assay, purified α_(llb)β₃ was addedto wells coated with either Cyr61 or Fisp12 (mCTGF) in the presence orabsence of inhibitors and binding was allowed to proceed for 3 hours at37° C. Unbound receptor was removed and the wells wee washed twice withHEPES-Tyrode's Buffer. The binding of purified α_(llb)β₃ to Cyr61 orFisp12/mCTGF immobilized onto microtiter wells was detected with¹²⁵I-mAb15.

Both activated and unactivated α_(llb)β₃ were indistinguishable onSDS-PAGE analysis as detected by silver staining. However, activatedα_(llb)β₃, but not the unactivated receptor, was capable of binding toimmobilized fibrinogen. Likewise, greater binding of activated versusunactivated α_(llb)β₃ to Fisp12/mCTGF and Cyr61 was observed. Incontrast, the background bindings of activated and unactivated α_(llb)β₃to control wells coated with BSA were similar. Thus, activated, but notunactivated, platelets adhered to Cyr61 and Fisp12/mCTGF.

To further characterize the interaction of α_(llb)β₃ with Fisp12/mCTGFand Cyr61, binding isotherms were determined for varying concentrationsof RGD-affinity purified α_(llb)β₃. These binding isotherms showed thatthe dose-dependent binding of activated α_(llb)β₃ to Fisp12mCTGF andCyr61 was saturable with half-saturation occurring at 15 nM and 25 nMα_(llb)β₃, respectively. Again, no significant binding of α_(llb)β₃ tocontrol BSA-coated wells was observed. To demonstrate the specificity ofthe interaction, inhibition studies were performed. As expected, thebinding of activated α_(llb)β₃ to Fisp12/mCTGF and Cyr61 wasspecifically blocked by RGDS but not by RGES. Furthermore, echistatinand the H₁₂ peptide also effectively inhibited α_(llb)β₃ binding tothese proteins. These findings are consistent with results obtained inthe platelet adhesion assay. Collectively, these functional andbiochemical data demonstrate that activated integrin α_(llb)β₃ is thereceptor mediating activation-dependent platelet adhesion to Cyr61 andFisp12/mCTGF.

Thus, another aspect of the invention is a method of screening formodulators of wound healing comprising the steps of: (a) contacting afirst activated platelet with a polypeptide of the CCN family, such asCyr61, and a suspected modulator; (b) further contacting a secondactivated platelet with the polypeptide of step (a); (c) measuring thebinding of the first activated platelet to the polypeptide; (d)measuring the binding of the second activated platelet to thepolypeptide; and (e) comparing the binding measurements of steps (d) and(e), thereby identifying a modulator of wound healing. Preferably, thewound healing involves the participation of platelet binding in theprocess of blood clotting. Also preferred are platelets presenting theα_(llb)β₃ integrin.

In addition to the above-described binding properties of members of theCCN family of proteins, antibody inhibition studies with anti-α_(M) andanti-β₃ antibodies have shown that Cyr61 binds to macrophages via yetanother integrin, the α_(M)β₂ integrin. Based on these results, it isexpected that mammalian CCN proteins, such as human or mouse Cyr61, willbind to the macrophages of mammals. It is also expected that Cyr61 willpromote the migration of macrophages, thus serving a role in attractingand retaining macrophages at the site of a wound. Consequently, Cyr61 isexpected to play a role in the inflammatory response of mammals, andmodulation of Cyr61 activity is expected to influence the inflammatoryresponse.

Yet another aspect of the invention is a method of screening formodulators of macrophage adhesion comprising the steps of: (a)contacting a first macrophage with a polypeptide of the CCN family, suchas Cyr61, and a suspected modulator; (b) further contacting a secondmacrophage with the polypeptide of step (a); (c) measuring the bindingof the first macrophage to the polypeptide; (d) measuring the binding ofthe second macrophage to the polypeptide; and (e) comparing the bindingmeasurements of steps (d) and (e), thereby identifying a modulator ofmacrophage adhesion.

EXAMPLE 33 Peptide Modulators of ECM Signaling Molecule Activity

Screening assays for modulators of cell adhesion are designed toidentify modulators (e.g., inhibitors or activators) of ECM signalingmolecule activities, such as Cyr61 activities, involved in angiogenesis,chondrogenesis, oncogenesis, cell adhesion, cell migration or cellproliferation. In developing a screening assay for mudulators of celladhesion, candidate peptide modulators were designed as described inExample 12. In particular, Cyr61 peptide fragments were designed withthe expectation that such peptides would modulate Cyr61 binding to anintegrin receptor such as the α₆β₁ integrin.

To facilitate success with the approach to peptide design described inExample 12, experiments were conducted to refine the location of domainsinvolved in one or more of the activities of Cyr61, such as angiogenicactivities. Initially, a deletion of mouse cyr61 that removed domain IVof the encoded Cyr61 (corresponding to amino acids 282-381 of SEQ IDNO:4) was generated. For a general description of Cyr61 domains 1-4, seeLau et al., Exp. Cell Research 248:44-57 (1999), incorporated herein byreference in its entirety. The deletion construct, cloned using aconventional Baculovirus system, was expressed and was subjected to arat corneal implant assay as generally described in Example 19, and theresults indicated that the truncated polypeptide induced angiogenesis.Further, the truncated polypeptide was subjected to an in vitroendothelial cell migration assay as described in Example 15, and thepolypeptide containing Cyr61 domains I, II and III induced cellmigration.

To further localize domains involved in angiogenesis, includingendothelial cell migration, the coding regions for domains I, II and IIIof Cyr61 were separately fused to a coding sequence for glutathioneS-transferase (GST), again using conventional technology. The individualfusion constructs were introduced into bacterial hosts, expressed, andpurified with glutathione columns, using conventional techniques. Celladhesion assays using endothelial cells and fibroblasts showed that, inisolation, only domain III (corresponding to amino acids 212-218 of SEQID NO:4) supported cell adhesion, and the fusion polypeptide containingthis domain, when immobilized, supported the adhesion of both restingendothelial cells and fibroblasts.

To localize the relevant domain still further, several overlappingpeptides were synthesized. The peptides, each having a sequence setforth in one of SEQ ID NOS:33-38, show significant similarity to an ECMsignaling molecule such as mouse Cyr61 (SEQ ID NO:2) or human Cyr61 (SEQID NO:4). These peptides were then separately tested for theircapacities to inhibit Cyr61 adhesion to endothelial cells orfibroblasts. Peptides were separately added at various concentrations toCyr61-coated wells containing endothelial cells or fibroblasts, asdescribed above. The peptide designated TSP1 (SEQ ID NO:33) inhibitedCyr61 adhesion to endothelial cells or fibroblasts at concentrations of25 μM or higher. None of the other peptides tested (SEQ ID NO:34-38)showed any inhibition of cell adhesion to Cyr61.

To determine if TSP1 was selectively inhibiting Cyr61 binding to α₅β₃ orα₆β₁, two integrins found on endothelial cells that bind Cyr61, specificmonoclonal antibody blocking studies were performed. These studiesinvolved the separate addition of monoclonal antibody GoH3 (α₆-specific,including α₆β₁; see Example 29) or LM609 (α₅β₃ specific; see Example 29)to cells prior to addition of the mixture to Cyr61-coated wells in thepresence or absence of the peptide under study. The results showed thatTSP1 inhibitied Cyr61 binding to the α₆β₁ integrin, a major Cyr61receptor active in resting, or unactivated, endothelial cells and infibroblasts. TSP1 did not inhibit Cyr61 binding to α_(V)β₃, a majorCyr61 receptor in activated endothelial cells.

Endothelial cells interacting with Cyr61 require integrin α_(V)β₃ forcell migration and cell survival. However, to form tubules in vitro in amatrigel, integrin α₆β₁ is required. Thus, the ability of TSP1 toinhibit tubule formation in vitro using a matrigel assay as described inExample 15 (and as known in the art, Davis et al., Exp. Cell Research216:113-123 (1995), incorporated herein by reference in its entirety)was performed. As expected, TSP1 inhibited tubule formation in thematrigel assay.

To confirm that the TSP1 peptide was inhibiting Cyr61 activity bydirectly interacting with the α₆β₁, integrin, TSP1 and the peptidescollectively having SEQ ID NOS:34-38 were used in the aforementionedcell adhesion assay in the absence of immobilized Cyr61. In theseassays, wells were coated with one of the peptides and the immobilizedpeptides were then separately exposed to either endothelial cells(resting or activated) or fibroblasts. The results showed that TSP1, byitself, supported adhesion of endothelial cells and fibroblasts. None ofthe other tested peptides (having SEQ ID NOS:34-38, collectively)supported cell adhesion in these assays. However, it is expected thatpeptides comprising sequences from domain II (von Willebrand Factordomain) of Cyr61 will inhibit the interaction between Cyr61 and theα_(V)β₃ integrin receptor, thereby inhibiting the participation ofendothelial cells in the process of angiogenesis. Such peptides areexpected to show at least 95%, and preferably 98%, similarity to asubsequence of the sequence set forth from amino acid 93 to amino acid211 of SEQ ID NO:4 using the comparison algorithm of Altschul et al.used for BLAST searching in the GenBank nucleotide database(http://www.ncbi.nlm.nih.gov/) with default settings in place. Peptidesexhibiting activity are expected to be at least seven amino acids inlength, with no upper bound, although the most economical peptides willprobably have no more than 20 amino acids.

This example establishes that peptide modulators of ECM signalingmolecules, such as mouse and human Cyr61 can be identified using the invitro angiogenesis and in vitro cell adhesion assays of the invention.The modulators themselves, showing promise as potential therapeutics fordiseases and conditions related to angiogenesis, chondrogenesis, andcell adhesion, and for oncogenesis, cell migration and cellproliferation, constitute another aspect of the invention.

One of ordinary skill in the art, apprised of the information disclosedin this application, could readily identify other peptides having Cyr61modulating activity by designing candidate peptides having sequencesimilarity to an ECM signaling molecule such as Cyr61. Sequencevariation is guided by a knowledge of conservative amino acidssubstituting as described above, peptide length may be varied betweenabout 8-50 amino acid residues. Further, peptide derivatives (e.g.,glycosylated, PEGylated, phosphorylated, may be used, as would be knownin the art.

Numerous modifications and variations in the practice of the inventionas illustrated in the above examples are expected to occur to those ofordinary skill in the art. Consequently, the illustrative examples arenot intended to limit the scope of the invention as set out in theappended claims.

1. An isolated monoclonal antibody that specifically binds to humanCyr61, wherein the antibody binds to amino acids 163-229 of SEQ ID NO:4.2. The antibody of claim 1, wherein the antibody binds to amino acids210-225 of SEQ ID NO:4.
 3. The antibody of claim 1 or 2, wherein theantibody is a humanized antibody.
 4. The antibody of claim 1 or 2,wherein the antibody is an antibody fragment.
 5. The antibody of claim 1or 2, wherein the antibody is a chimeric antibody.
 6. The antibody ofclaim 1 or 2, wherein the antibody is a CDR-grafted antibody.